JP2014530598A - Polypeptide having protease activity and polynucleotide encoding the same - Google Patents

Abstract

The present invention relates to an isolated polypeptide having protease activity and an isolated polynucleotide encoding the polypeptide. The invention also relates to nucleic acid constructs comprising polynucleotides, vectors and host cells, as well as methods for producing and using the polypeptides.

Description

Reference to Sequence Listing This application contains a sequence listing in computer readable form, which is incorporated herein by reference.

  The present invention relates to isolated polypeptides having protease activity and isolated nucleic acid sequences encoding proteases. The invention also relates to nucleic acid constructs comprising nucleic acid sequences, vectors and host cells (such as plant and animal cells) and methods for producing and using proteases, particularly the use of proteases in animal feeds and detergents.

  The present invention provides polypeptides having protease activity and polynucleotides encoding the polypeptides.

  In the detergent industry, different enzymes have been used in detergent formulations for over 30 years and the most commonly used enzymes are proteases, amylases, each adapted to remove various types of soils. And lipase. In addition to enzymes, detergent compositions typically include complex combinations of formulation ingredients. For example, most cleaning products include surfactant systems, bleaching agents or builders. Despite the complexity of current detergents, there is still a need to develop new detergent compositions that include new enzymes and / or enzyme blends.

  Traditionally, washing is done at high temperatures, and well-known detergents are chosen to be carried out at higher temperatures.

  Increasing emphasis on improving the cleaning process to further reduce the environmental burden, reduces the cleaning time, pH and temperature, and the amount of detergent ingredients that may impact the environment globally The tendency to reduce is seen.

  Accordingly, there is a need for laundry at lower temperatures, and hence a need for detergent proteases that have high performance at low temperatures.

  It is understood that proteins are essential nutrients for animals and humans in the use of proteases in animal feed (in vivo) and / or in the use of proteases described above for processing plant proteins (in vitro). . Most livestock and many humans ingest the necessary proteins from vegetable protein sources. Important plant protein sources are, for example, oilseed crops, legumes and cereals.

  For example, if soy meal is included in the diet of monogastric animals such as pigs and poultry, a significant proportion of soybean meal solids is not efficiently digested (pigs, growing pigs, and broilers, laying hens and The apparent ileal protein digestibility in poultry such as roosters is only around 80%).

  The animal gastrointestinal tract is composed of a series of segments, each representing a different pH environment. In monogastric animals such as pigs and poultry and a variety of fish, the stomach exhibits a low acidic pH of 1-2 and a strong acidic pH, whereas the intestine has a more neutral pH in the region of pH 6-7. Show. Poultry has a stomach in front of the stomach in addition to the gastrointestinal tract, the pH of which is most often measured by ingested feed and is therefore typically in the pH 4-6 range. Protein digestion with proteases can be performed along the entire digestive tract if the protease is active and survives the conditions in the digestive tract. Accordingly, proteases that are highly acid stable in the gastric environment while being effectively active at a wide range of physiological pH in the subject animal are particularly desirable.

  Also, animal feed is often prepared in pelletized form, where the feed stream is subjected to a pelletizing process. Therefore, it is also desirable that the protease used in animal feed retains activity after exposure to the feed stream treatment.

  The use of proteases in animal feed to improve the digestion of proteins in the feed is known. WO 95/28850 discloses a combination of phytase and one or more microbial proteolytic enzymes to improve the solubility of plant proteins. WO 01/58275 discloses the use of a subtilisin family of acid stable proteases in animal feed. WO 01/58276 discloses in animal feeds acid stable proteases related to proteases from Nocadiopsis sp. NRRL 18262 (10R protease), as well as proteases from Norcadiopsis alba DSM 14010. Disclose use. WO 04/072221, WO 04/111220, WO 04/111223, WO 05/035747, and 05/123911 are incorporated into 10R protease. Related proteases and their use in animal feed are disclosed. WO 04/072279 discloses the use of other proteases.

  WO 04/034776 discloses the use of subtilisin / keratinase, PWD-1, from B. licheniformis in poultry feed. WO 04/077960 discloses a method for increasing fodder or grain digestibility in ruminants by applying bacterial or fungal proteases.

  Products containing proteases that are commercially available for use in animal feed include RONOZYME® ProAct (DSM NP / Novozymes), Axtra® (Danisco), Avizyme® (Danisco), Porzyme (R) (Danisco), Allzyme (TM) (Alltech), Versozyme (R) (BioResources, Int.), Poultrygrow (TM) (Jefo), and Cibenza (R) DP100 (Novus).

The present invention is:
(A) at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% relative to the mature polypeptide of SEQ ID NO: 2 A polypeptide having a sequence identity of at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%;
(B) at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89% relative to the mature polypeptide encoding the sequence of SEQ ID NO: 1 Having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity A polypeptide encoded by the polynucleotide;
(C) a variant of the mature polypeptide of SEQ ID NO: 2 comprising substitutions, deletions and / or insertions at one or more (eg several) positions; and (d) having protease activity (a) , (B) or (c) relates to an isolated polypeptide having protease activity selected from the group consisting of fragments of the polypeptide.

  The invention also relates to an isolated polynucleotide encoding a polypeptide of the invention; a nucleic acid construct; a recombinant expression vector; a recombinant host cell comprising the polynucleotide; and a method for producing the polypeptide.

  The invention further relates to the use of the protease of the invention in animal feeds and detergents, methods for producing animal feed compositions and detergent compositions, and animal feed compositions and detergent compositions.

  The present invention also includes a polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 29 of SEQ ID NO: 2, each operably linked to a gene encoding a protein, SEQ ID NO: 2 A polynucleotide encoding a propeptide comprising or consisting of amino acids 30 to 188, or a polynucleotide encoding a signal peptide and propeptide comprising or consisting of amino acids 1 to 188 of SEQ ID NO: 2, respectively Nucleic acid constructs comprising polynucleotides, expression vectors and recombinant host cells; and methods for producing proteins.

FIG. 5 shows the activity of S1 protease from Saccharothrix australiensis on soybean-corn meal compared to 10R protease.

Polypeptides with defined protease activity Polypeptides with protease activity, ie proteases, are also sometimes referred to as peptidases, proteinases, peptide hydrolases, or proteolytic enzymes. The protease may be either an exo-type that hydrolyzes the peptide starting from either end, or an endo-type that acts inside the polypeptide chain (endopeptidase). Endopeptidases are active against peptide substrates blocked at the N- and C-termini, which is related to the specificity of the protease of interest.

  The term “protease” is defined herein as an enzyme that hydrolyzes peptide bonds. This definition of protease also applies to the protease portion of the terms “parent protease” and “protease variant” as used herein. The term “protease” encompasses all enzymes belonging to the EC 3.4 enzyme group (including each of its 13 subgroups). EC number refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, Eur. J. et al. Bio-chem. 1994, 223, 1-5; Eur. J. et al. Biochem. 1995, 232, 1-6; Eur. J. et al. Biochem. 1996, 237, 1-5; Eur. J. et al. Biochem. 1997, 250, 1-6; and Eur. J. et al. Biochem. Increment 1-5 published in 1999, 264, 610-650, respectively. Scientific names are regularly supplemented and updated; see, for example, World Wide Web (WWW) site http: // www. chem. qmw. ac. uk / ibubmb / enzyme / index. See html.

Proteases of the invention and proteases for use in accordance with the invention are described in Biochem. J. et al. 290: 205-218 (1993) and the MEROPS protease database, publication, 9.4 (www.merops.ac.uk), the following:
(A) EC 3.4.21. A protease belonging to the enzyme group; and / or (b) selected from the group consisting of serine proteases of the peptidase family S1. The database is available from Rawlings, N .; D. Barrett, A .; J. et al. & Bateman, A.A. (2010) MEROPS: the peptidase database. Nucleic Acids Res 38, D227-D233.

  To determine whether a given protease is a serine protease or an S1 family protease, reference is made to the aforementioned Handbook and the principles set forth therein. Such determinations can be made for any type of protease, including naturally occurring proteases, or wild type proteases; or genetically engineered or synthesized proteases.

  S1 family peptidases contain three catalytic residues in the order His, Asp, Ser. Mutation of any of the catalytic tri-residue amino acids results in a change or loss of enzyme activity. The catalytic triad amino acids of the S1 protease from Saccharothrix australiensis (SEQ ID NO: 2) are probably at positions His-32, Asp-56 and Ser-136.

  Protease activity may be measured using any assay that uses a substrate that contains a peptide bond related to the specificity of the protease of interest. The assay pH and assay temperature are similarly adjusted for the protease of interest. Examples of assay pH values are pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Examples of assay temperatures are 15, 20, 25, 30, 35, 37, 40, 45, 50, 55, 60, 65, 70, 80, 90, or 95 ° C. An example of a protease substrate is casein, such as Azure-Crosslinked Casein (AZCL-casein), or suc-AAPF-pNA. Examples of suitable protease assays are described in the Examples section.

  Protease activity: The term “protease activity” means proteolytic activity (EC 3.4.21.) That catalyzes hydrolysis of an amide bond or protein by hydrolysis of peptide bonds that bind amino acids in a polypeptide chain. To do. A number of assays for determining protease activity are available in the art. For the purposes of the present invention, protease activity can be determined using Protazyme AK tablets (cross-linked and stained casein; manufactured by Megazyme) as described in the examples of this application. The polypeptide of the invention is at least 20% of the mature polypeptide of SEQ ID NO: 2, such as at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or Have at least 100% protease activity.

  Allelic variant: The term “allelic variant” means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation occurs naturally through mutations and can be caused by polymorphisms in a population. A genetic mutation can be silent (no change in the encoded polypeptide) or a polypeptide having an altered amino acid sequence can be encoded. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

  Catalytic domain: The term “catalytic domain” means a region of an enzyme that contains the catalytic mechanism of the enzyme.

  cDNA: The term “cDNA” refers to a DNA molecule that can be prepared by reverse transcription from a mature spliced mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. Early primary RNA transcripts are precursors of mRNA that are processed through a series of steps that involve splicing before appearing as mature spliced mRNA.

  Cleaning composition: The terms “cleaning composition” and “cleaning formulation” refer to items to be cleaned, such as fabrics, carpets, tableware including glass utensils, contact lenses, tiles, zinc plates, floors and table surfaces, etc. Refers to a composition used to remove unwanted compounds from hard surfaces, hair (shampoo), skin (soap and cream), teeth (mouthwash, toothpaste), and the like. This term refers to the type of cleaning composition and product desired (eg, liquid, gel) as long as the composition is compatible with the proteases of the invention used in the composition, and other enzymes. , Granules, or spray compositions). The particular choice of cleaning composition material is facilitated by considering the surface to be cleaned, the item or fabric, and the desired composition form for the cleaning conditions in use. These terms further refer to any composition suitable for cleaning, bleaching, disinfecting and / or sterilizing any object and / or surface. These terms include, but are not limited to, detergent compositions (eg, liquid and / or solid laundry detergents and fine fabric detergents; hard for glass, wood, ceramic and metal counter surfaces and windows, etc. It is intended to include surface cleaning formulations; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and fabric and laundry pre-stain removers and dishwashing detergents.

  Coding sequence: The term “coding sequence” means a polynucleotide that directly defines the amino acid sequence of a polypeptide. The boundaries of a coding sequence are generally determined by an open reading frame, which starts with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

  Control sequence: The term “control sequence” means a nucleic acid sequence necessary for the expression of a polynucleotide encoding the mature polypeptide of the present invention. Each regulatory sequence may be native (ie, derived from the same gene) or foreign (ie, derived from a different gene) to the polynucleotide encoding the polypeptide, or native to each other Or it may be a foreign one. Such control sequences include, but are not limited to, leaders, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, the control sequences include a promoter and transcriptional and translational termination signals. The control sequence may include a linker for the purpose of introducing a specific restriction site that facilitates linking the control region to the coding region of the polynucleotide encoding the polypeptide.

  Detergent composition: The term “detergent composition” means, unless otherwise specified, a general or heavy detergent in particulate or powder form, especially a cleaning detergent; in particular a so-called heavy liquid (HDL) type liquid, General detergents in gel or paste form; liquid detergents for fine fabrics; hand dishwashing or light dishwashing agents, especially those of high foaming type; various tablets, granules, liquids and rinses for home and industrial use Dishwashing chemicals for machines including auxiliary types; antibacterial hand washing types, cleaning bars, mouthwashes, denture cleaners, car or carpet shampoos, liquid cleaners and disinfectants including bathroom cleaners; hair shampoos and hair rinses; shower gels, bubble baths Metal cleaners; and bleach additives and “stain removers”; Click including cleaning aids such as (stain-stick) "or pre-processing type. The terms “detergent composition” and “detergent formulation” are used in reference to mixtures intended for use in cleaning media for cleaning contaminated objects. In some embodiments, the term is used to refer to the washing of fabrics and / or clothes (eg, “laundry detergent”). In an alternative embodiment, the term refers to other detergents, such as those used to clean dishes, cutlery, etc. (eg, “dishwashing detergent”). It is not intended that the present invention be limited to any particular detergent formulation or composition. This term is used in addition to the protease according to the invention, for example, surfactants, builders, chelating agents or chelating agents, bleach systems or bleach components, polymers, fabric conditioners, foam enhancers, soap foams. Inhibitors, dyes, fragrances, anti-fading agents, optical brighteners, bactericides, bactericides / fungicides, contaminant suspensions, corrosion inhibitors, enzyme inhibitors or stabilizers, enzyme activators, transferases It is intended to encompass detergents containing hydrolytic enzymes, oxidoreductases, bluing and fluorescent dyes, antioxidants, and solubilizers.

  Dishwashing composition: The term “dishwashing composition” refers to any form of composition for cleaning hard surfaces. The present invention is not limited to any particular type of dishwashing composition or particular detergent.

  Enzyme detergency benefit: As used herein, the term "enzyme detergency benefit" or "detergency" refers to the beneficial effect that an enzyme can add to a detergent compared to the same detergent without an enzyme. Defined. An important detergency benefit that can be brought about by the enzyme is soil removal with little or no visible contaminants after washing and / or cleaning; liberated in the washing process, an effect also called anti-redeposition prevention Preventing or reducing the re-adhesion of contaminants; the whiteness of fabrics that were originally white but became grayish or yellowish after repeated use and cleaning, an effect also known as whitening Is a complete or partial recovery. Textile care benefits, which are not directly related to catalyst soil removal or prevention of reattachment of contaminants, are also important for enzyme detergency benefits. Examples of the benefits of handling such fabrics are prevention or reduction of transfer from one fabric to another, or other parts of the same fabric, also called anti-transfer or anti-fouling prevention; Removal of protruding or broken fibers from the fabric surface to reduce pilling, also called anti-pills, or removal of existing pills or fluff; improved fabric flexibility; clarification of fabric color; And removal of particulate contaminants clogged in fabric or clothing fibers. Enzymatic bleaching is an additional enzyme detergency benefit, where catalytic activity is generally used to catalyze the formation of bleaching components such as hydrogen peroxide or other peroxides.

  Expression: The term “expression” includes, but is not limited to, any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification and secretion.

  Expression vector: The term "expression vector" includes a polynucleotide that encodes a polypeptide and includes linear or circular DNA molecules that are operably linked to regulatory sequences that effect its expression.

  Fabric: The term “fabric” encompasses any fabric material. The term is therefore intended to encompass garments as well as fabrics, yarns, fibers, nonwoven materials, natural materials, synthetic materials, and any other fabric material.

  Fragment: The term “fragment” means a polypeptide having one or more (several) amino acids deleted from the amino and / or carboxyl terminus of the mature polypeptide or domain; Has protease activity.

  Hard surface cleaning: As used herein, “hard surface cleaning” is defined as cleaning a hard surface, where a hard surface is a floor, table, wall, roof, etc., as well as automobiles (car cleaners) and dishes (dishwashing) The surface of a hard object such as an agent. Dishwashing includes, but is not limited to, cleaning of plates, cups, glass tableware, balls, and tableware such as spoons, knives, forks, cutlery, ceramics, plastics, metals, ceramics, glass and acrylic products. Can be mentioned.

  High stringency conditions: The term “high stringency conditions” refers to a probe of at least 100 nucleotides in length, followed by a standard Southern blotting method of 12-24 hours, 42 ° C., 5 × SSPE, 0.3% Means SDS, 200 microgram / ml fragment processing and modified salmon sperm DNA, and pre-hybridization and hybridization with 50% formamide. The carrier material is finally washed three times for 15 minutes each at 65 ° C. using 2 × SSC, 0.2% SDS.

  Host cell: The term “host cell” means any cell type that is sensitive to transformation, transfection, transduction, etc., with a nucleic acid construct or expression vector comprising a polynucleotide of the invention. The term “host cell” encompasses any progeny of a parent cell that is not the same as the parent cell due to mutations that occur during replication.

  Improved cleaning performance: The term “improved cleaning performance” as used herein refers to a change in cleaning performance of a protease variant, for example, due to increased soil removal relative to the cleaning performance of the parent protease variant. (Variants) Defined as enzymes (also including enzymes, not necessarily mutants, but also backbone blends, combinations with specific cleaning compositions, etc.). The term “washing performance” includes not only washing but also washing performance in dishwashing, for example.

  Isolated: The term “isolated” means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated material include (1) any non-natural material, (2) one or more of the natural constituents associated in nature, but not limited to Any substance containing any enzyme, variant, nucleic acid, protein, peptide or cofactor that is at least partially removed from all; (3) any artificially modified material found in nature Or (4) any substance modified by an increase in the amount of the substance relative to other components that are naturally related (eg, multiple copies of the gene encoding the substance; the gene encoding the substance; Use of a stronger promoter than the one associated in nature). Isolated material may be present in the fermentation broth sample.

  Low stringency conditions: The term “low stringency conditions” refers to a probe of at least 100 nucleotides in length, following a standard Southern blotting method of 12-24 hours, 42 ° C., 5 × SSPE, 0. Means prehybridization and hybridization with 3% SDS, 200 microgram / ml fragment treatment and modified salmon sperm DNA, and 25% formamide. The carrier material is finally washed three times for 15 minutes each at 50 ° C. using 2 × SSC, 0.2% SDS.

  Mature polypeptide: The term “mature polypeptide” means a polypeptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. To do. In one aspect, the mature polypeptide is amino acids 189-374 of SEQ ID NO: 2, based on amino acid sequencing using Edman degradation chemistry. It is known that host cells can produce a mixture of two further different mature polypeptides (ie, comprising different C-terminal and / or N-terminal amino acids).

  Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” means a polynucleotide that encodes a mature polypeptide having protease activity. In one aspect, the mature polypeptide coding sequence is nucleotides 665 to 1222 of SEQ ID NO: 1.

  Moderate stringency conditions: The term “moderate stringency conditions” refers to a probe of at least 100 nucleotides in length, following a standard Southern blotting method of 12-24 hours, 42 ° C., 5 × SSPE, 0. Means prehybridization and hybridization with 3% SDS, 200 microgram / ml fragment treatment and modified salmon sperm DNA, and 35% formamide. The carrier material is finally washed three times for 15 minutes each at 55 ° C. using 2 × SSC, 0.2% SDS.

  Medium-high stringency conditions: The term “medium-high stringency conditions” refers to a probe that is at least 100 nucleotides in length, followed by a standard Southern blotting method of 12-24 hours at 42 ° C., 5 × SSPE. , 0.3% SDS, 200 microgram / ml fragment treatment and modified salmon sperm DNA, and prehybridization and hybridization with 35% formamide. The carrier material is finally washed three times for 15 minutes each at 60 ° C. using 2 × SSC, 0.2% SDS.

  Nucleic acid construct: The term “nucleic acid construct” means a nucleic acid molecule, either single stranded or double stranded, which is isolated from a natural gene or otherwise non-naturally occurring. It will be modified to contain segments of nucleic acid in a manner that is likely to be synthetic or it will contain one or more regulatory sequences.

  Operably linked: The term “operably linked” is a structure that places a control sequence in an appropriate position relative to the coding sequence of a polynucleotide, such that the control sequence directs expression of the coding sequence. Means.

  Sequence identity: The relationship between two amino acid sequences or between two nucleotide sequences is represented by the parameter “sequence identity”.

For the purposes of the present invention, the degree of sequence identity between two amino acid sequences is preferably determined from the version 5.0.0 or later EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000). , Trends Genet. 16: 276-277), the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453). The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5 and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Equivalent residue × 100) / (alignment length−total number of gaps in alignment)

For the purposes of the present invention, the degree of sequence identity between two deoxyribonucleotide sequences is preferably determined by the EMBOSS package from version 5.0.0 or later (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al. , 2000) using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970 described above). The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5 and an EDNAFULL (EMBIOSS version of NCBI NUC 4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Equivalent deoxyribonucleotide x 100) / (alignment length-total number of gaps in the alignment)

  Subsequence: The term “subsequence” means a polynucleotide having one or more (eg, several) nucleotides deleted from the 5 ′ and / or 3 ′ end of the mature polypeptide coding sequence. Where the subsequence encodes a fragment having protease activity.

  Substantially pure polynucleotide: The term “substantially pure polynucleotide” is free of other foreign or unwanted nucleotides and is suitable for use in genetically engineered polypeptide production systems. Means a form of polynucleotide preparation. Thus, a substantially pure polynucleotide is up to 10%, up to 8%, up to 6%, up to 5%, up to 5% by weight of other polynucleotide material that is originally or recombinantly related. 4 wt%, up to 3 wt%, up to 2 wt%, up to 1 wt% and up to 0.5 wt%. However, substantially pure polynucleotides can include natural 5'- and 3'-untranslated regions such as promoters and terminators. Preferably, the polynucleotide is at least 90% pure by weight, eg, at least 92% pure, at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% purity, at least 99% purity and at least 99.5% purity. The polynucleotide of the present invention is preferably in a substantially pure form.

  Substantially pure polypeptide: The term “substantially pure polypeptide” refers to a maximum of 10%, a maximum of 8%, a maximum of 6%, a maximum of 5%, and a maximum of 4% by weight. Means preparations containing up to 3% by weight, up to 2% by weight, up to 1% by weight and up to 0.5% by weight of other polypeptide materials originally or recombinantly related To do. Preferably, the polypeptide is at least 92% pure, for example at least 94% pure, at least 95% pure, at least 96% pure, based on the total weight of polypeptide material present in the preparation. At least 97% purity, at least 98% purity, at least 99%, at least 99.5% purity and 100% purity. The polypeptide of the present invention is preferably in a substantially pure form. This can be accomplished, for example, by preparing the polypeptide by well-known recombinant methods or by classical purification methods.

  Fabric: The term “fabric” refers to any fabric material such as yarn, yarn intermediate, fiber, nonwoven material, natural material, synthetic material, and any other fabric material, fabrics made from these materials, As well as products made from fabric (eg, clothing and other articles). The fabric or fabric may be in the form of a knit, woven, denim, non-woven, felt, yarn and toweling. The fabric is a natural cellulosic material including cotton, flax / linen, jute, ramie, sisal or coir, or artificial cellulose containing viscose / rayon, ramie, cellulose acetate fibers (tricellular), lyocell or blends thereof It may be a cellulosic material such as a system material (for example, a material derived from wood pulp). The fabric or fabric can also be a non-cellulosic material such as natural polyamides such as wool, camel, cashmere, mohair, rabbit and silk, or synthetic polymers such as nylon, aramid, polyester, acrylic, polypropylene and spandex / elastan, or These blends and blends of cellulosic and non-cellulosic fibers may be used. Examples of blends are cotton and / or rayon / viscose and wool, synthetic fibers (eg polyamide fibers, acrylic fibers, polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers), And blends with one or more accompanying materials such as cellulose-containing fibers (eg, rayon / viscose, ramie, flax / linen, jute, cellulose acetate fiber, lyocell). The fabric may be a conventional washable laundry, such as a dirty home laundry, for example. Where the term fabric or garment is used, it shall also include the term broader fabric.

  Dough care benefits: “Dough care benefits” are not directly related to catalytic soil removal or prevention of redeposition of contaminants, but are also important for enzyme cleansing benefits. Examples of benefits from such fabric care include the following: prevention or reduction of transfer from one fabric to another or to another part of the same fabric (transfer suppression or vice versa). (Also called contamination prevention); removal of protruding or broken fibers from the fabric surface to reduce pilling (action also called anti-pilling) or removal of existing pilling or fluff; increased fabric flexibility, fabric Clarification of color and removal of particulate contaminants trapped in fabric fibers. Enzymatic bleaching is an additional enzyme detergency benefit, where catalytic activity is generally used to catalyze the formation of bleaching components such as hydrogen peroxide or other peroxides or other types of bleaching agents. .

  Variant: The term “variant” means a polypeptide having protease activity that includes modifications (ie, substitutions, insertions and / or deletions) at one or more (eg, several) positions. A substitution means the replacement of an amino acid at a position with a different amino acid; a deletion means the removal of an amino acid at a position; and an insertion is an immediately adjacent amino acid at a position that immediately follows 1 It means adding one or more (eg several) amino acids, eg 1-5 amino acids. A variant of the invention has a maturation of SEQ ID NO: 2 of at least 20%, such as at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100% Protease activity of type polypeptide.

  Very high stringency conditions: The term “very high stringency conditions” refers to a standard Southern blotting method of 12-24 hours for probes of length at least 100 nucleotides, 42 ° C., 5 × SSPE, 0. Means prehybridization and hybridization with 3% SDS, 200 microgram / ml fragment treatment and modified salmon sperm DNA, and 50% formamide. The carrier material is finally washed three times for 15 minutes each at 70 ° C. using 2 × SSC, 0.2% SDS.

  Very low stringency conditions: The term “very low stringency conditions” refers to a probe that is at least 100 nucleotides in length, following a standard Southern blotting method of 12-24 hours, 42 ° C., 5 × SSPE, Means prehybridization and hybridization with 0.3% SDS, 200 microgram / ml fragment treatment and modified salmon sperm DNA, and 25% formamide. The carrier material is finally washed 3 times for 15 minutes each at 45 ° C. using 2 × SSC, 0.2% SDS.

  Cleaning performance: The term “cleaning performance” is used as the enzyme's ability to remove dirt present on the object to be cleaned, for example during cleaning or hard surface cleaning.

  Whiteness: The term “whiteness” is defined herein as a broad term that has different meanings for different customers in different fields. The loss of whiteness can be due, for example, to graying, yellowing, or removal of optical brightener / colorant. Graying and yellowing can occur due to stain redeposition, body stains, colorants or transfer from iron and iron ions, and the like. Whiteness may include one or more items from the list listed below: dye or dyeing effect; incomplete soil removal (eg, body stains, sebum, etc.); reattachment (grayed object) , Yellowing, or other discoloration) (removed soil recombined with another portion of soiled or unstained fabric); chemical changes in the fabric in use; and color clarification or whitening .

Polypeptides with Protease Activity In one embodiment, the invention provides at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% Maturation of SEQ ID NO: 2 with at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity It relates to an isolated polypeptide having a type polypeptide and having protease activity. In one aspect, the polypeptide comprises a mature polypeptide of SEQ ID NO: 2 and no more than 20, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, It differs by 14, 15, 16, 17, 18 or 19 amino acids.

  The polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof having protease activity. In another embodiment, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 2. In other embodiments, the polypeptide comprises or consists of amino acids 189-374 of SEQ ID NO: 2.

  In another embodiment, the invention may be used under very low stringency conditions, low stringency conditions, moderate stringency conditions, moderate-high stringency conditions, high stringency conditions, or very high stringency conditions. , Hybridizing to the mature polypeptide coding sequence of SEQ ID NO: 1 or its full-length complement (Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York). It relates to an isolated polypeptide having protease activity encoded by a polynucleotide.

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, and the polypeptide of SEQ ID NO: 2 or a fragment thereof is a DNA encoding a polypeptide having protease activity from different genus or species strains according to methods well known in the art. Can be used in the design of nucleic acid probes to identify and clone. In particular, such probes can be used for hybridization with genomic DNA or cDNA of the cells of interest after standard Southern blotting to identify and isolate the corresponding genes involved. It is. Such probes can be considerably shorter than the entire sequence, but should be, for example, at least 25, at least 35, or at least 70 and at least 15 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, eg, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides or at least 900 nucleotides in length. That's it. Both DNA and RNA probes can be used. The probe is typically labeled to detect the corresponding gene (eg, with ³² P, ³ H, ³⁵ S, biotin or avidin). Such probes are encompassed by the present invention.

  Genomic DNA or cDNA libraries prepared from such other strains can be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having protease activity. Genome or other DNA from such other strains can be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from these libraries or isolated DNA can be transferred and immobilized on nitrocellulose or other suitable carrier material. Carrier material is used for Southern blotting to identify clones or DNA that hybridize to SEQ ID NO: 1 or 3 or subsequences thereof.

  For the purposes of the present invention, hybridization refers to (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQ ID NO: 1, under very low to very stringent conditions; Long complement; or (iv) means that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to its subsequence. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected, for example, by X-ray film or any other detection means known in the art.

  In one aspect, the nucleic acid probe is nucleotides 101 to 1405, nucleotides 188 to 1222, nucleotides 665 to 1222, or nucleotides 800 to 1200 of SEQ ID NO: 1. In other embodiments, the nucleic acid probe is a polynucleotide encoding the polypeptide of SEQ ID NO: 2; a mature polypeptide thereof; or a fragment thereof. In another embodiment, the nucleic acid probe is SEQ ID NO: 1.

  In another embodiment, the invention provides at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 relative to the mature polypeptide coding sequence of SEQ ID NO: 1. %, At least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% It relates to an isolated polypeptide having protease activity encoded by a polynucleotide having sequence identity.

  In another embodiment, the invention relates to a variant of the mature polypeptide of SEQ ID NO: 2, comprising substitutions, deletions and / or insertions at one or more (eg, several) positions. In one embodiment, the number of amino acid substitutions, deletions and / or insertions introduced into the mature polypeptide of SEQ ID NO: 2 is 20 or less, eg 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19. Amino acid mutations may be of secondary nature, ie, conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1-30 amino acids Small amino- or carboxyl-terminal extensions such as amino-terminal methionine residues; small linker peptides of 20-25 or fewer residues; or mutations or other such as polyhistidine sequences, antigenic epitopes or binding domains It may be a small extension that facilitates purification by changing its function.

  Examples of conservative substitutions are basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids ( Within the group of phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art, see, for example, H.P. Neuroth and R.M. L. Hill, 1979, In, The Proteins, Academic Press, New York. Frequent substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly.

  Alternatively, the amino acid mutation is of a nature such that the physicochemical properties of the polypeptide are altered. For example, amino acid mutations can be to improve the thermal stability of the polypeptide, alter substrate specificity, and change the optimal pH.

  Essential amino acids in a polypeptide can be identified according to techniques known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). . In the latter technique, a single alanine mutation is introduced into every residue in the molecule and the resulting mutant molecule is tested for protease activity to identify amino acid residues that are important for the activity of the molecule. Is done. Also, Hilton et al. 1996, J. MoI. Biol. Chem. 271: 4699-4708. The active site or other biological interaction of the enzyme is determined by techniques such as nuclear magnetic resonance, crystal structure analysis, electron diffraction or photoaffinity labeling, combined with putative contact site amino acid mutations. It can also be determined by physical analysis of the structure. For example, de Vos et al. , 1992, Science 255: 306-312; Smith et al. , 1992, J. et al. Mol. Biol. 224: 899-904; Wlodaver et al. , 1992, FEBS Lett. 309: 59-64. Essential amino acid identities can also be inferred from alignments with related polypeptides. In the polypeptide of the present invention, the essential amino acids forming the catalytic triad are identified as amino acids corresponding to His-220, Asp-244 and Ser-324 in SEQ ID NO: 2 by alignment with the amino acid sequence of 10R protease. Has been.

  Single or multiple amino acid substitutions, deletions, and / or insertions are described in Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-1156; WO 95/17413; or known mutagenesis methods, such as those disclosed by WO 95/22625, recombination methods, and / or shuffling. Method and subsequent related screening methods can be used and tested. Other methods that can be used include error-prone PCR, phage display (eg, Lowman et al., 1991, Biochemistry 30: 10832-10837; US Pat. No. 5,223,409; International Publication). No. 92/06204) and region-specific mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

  Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned mutagenic polypeptides expressed by host cells (Ness et al., 1999, Nature). Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cell and readily sequenced using standard methods in the art. With these methods, the importance of individual amino acid residues in a polypeptide can be readily determined.

  A polypeptide can be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or C-terminus of a region of another polypeptide.

  The polypeptide may be a fusion polypeptide or a cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or C-terminus of the polypeptide of the invention. A fusion polypeptide is generated by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the invention. Techniques for generating fusion polypeptides are known in the art and link the coding sequences encoding the polypeptides so that they are in frame and the expression of the fusion polypeptide is under the control of the same promoter and terminator. Includes steps. Fusion polypeptides may also be constructed using intein technology in which a fusion polypeptide is formed after translation (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science). 266: 776-779).

  The fusion polypeptide can further include a cleavage site between the two polypeptides. When the fusion protein is secreted, this site is cleaved to release the two polypeptides. Examples of cleavage sites include, but are not limited to, Martin et al. , 2003, J. et al. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al. , 2000, J. et al. Biotechnol. 76: 245-251; Rasmussen-Wilson et al. , 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al. , 1995, Biotechnology 13: 498-503; and Contreras et al. 1991, Biotechnology 9: 378-381; Eaton et al. , 1986, Biochemistry 25: 505-512; Collins-Racie et al. , 1995, Biotechnology 13: 982-987; Carter et al. 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

Embodiments In certain embodiments of the invention, the protease of the invention exhibits beneficial thermal properties such as thermal stability, steam stability, pH characteristics such as acid stability, and the like. Embodiments of the present invention are isolated having improved protease activity relative to protease 10R at 37 ° C-60 ° C, eg, 37 ° C-50 ° C, or 37 ° C, 50 ° C or 60 ° C. It is a polypeptide.

Acid / Alkaline Properties In certain embodiments of the present invention, the proteases of the present invention exhibit beneficial properties related to pH, such as acid stability. The stability of the protease at low pH is beneficial because the protease can retain activity in the intestine after passing through the stomach. The stability of proteases at high pH is beneficial for cleaning and washing because detergent compositions often have an alkaline pH. In one embodiment of the invention, the protease retains> 90% activity after 2 hours at pH 3 as determined using the method described in Example 4. In another embodiment of the invention, the protease retains> 90% activity after 2 hours at pH 9 as determined using the method described in Example 4.

  The present invention provides polypeptides having protease activity and polynucleotides encoding the polypeptides. The protease of the present invention is a serine protease of peptidase family S1. The proteases of the present invention exhibit surprising pH properties, particularly pH stability, which make them interesting candidates for use in animal feeds and / or detergents.

Cleaning performance In certain embodiments of the invention, the protease of the invention exhibits beneficial cleaning performance. S1 protease from Saccharothrix australiensis is more effective at removing soils than detergents that do not contain any protease. S1 protease from Saccharothrix australiensis is effective in removing blood and egg stains even at 20 ° C.

Feed Use The protease of the present invention is active against Suc-Ala-Ala-Pro-Phe-pNA within a wide range of pH 4 to 11, exhibiting particularly high activity in the range of pH 6 to 11, pH 3 to It is active against feed-related soy meal-corn meal substrate within a broad physiological pH range of 7 and retains more than 80% activity after 2 hours at a low pH of 2. Therefore, the S1 protease derived from Saccharothrix australiensis of the present invention is suitable for various feed applications.

Source of polypeptide having protease activity The polypeptide having protease activity of the present invention can be obtained from microorganisms of any genus. For the purposes of the present invention, the term “obtained from”, as used herein, refers to a polypeptide encoded by a polynucleotide by source or by source in relation to a given source. Is produced by the inserted line. In one embodiment, the polypeptide obtained from a given source is secreted extracellularly.

  The polypeptide can be a bacterial polypeptide. For example, the polypeptide may have Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Lactococcus, having protease activity. Gram-positive bacterial polypeptides such as Bacillus (Oceanobacillus), Staphylococcus, Streptococcus, Saccharothrix or Streptomyces polypeptides, or Gram-positive bacterial P. Genus (Campyloba) ter), E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas m, Pseudomonas m It can be a gram-negative bacterial polypeptide such as a Salmonella or Ureaplasma polypeptide.

  In one aspect, the polypeptide is an Actinobacteria, such as Actinomycetes, or Pseudonocardineae, or Pseudonocardiaceae or Pseudonocardiaceae, It is a protease derived from a cell of the genus Saccharothrix. In another embodiment, the polypeptide is a Saccharothrix australiansis polypeptide.

  The strains of these species can be found in the American Type Culture Collection (ATCC), the German Microbial Cell Culture Collection (DSMZ: Deutsche Samplung von Mikraorganisund und Zellkulturen GmbH: Vs. ), And many others such as the Agricultural Research Bureau Patent Culture Service Patent Culture Collection, Northern Research Center (NRRL) Are readily available to the public in microorganisms save institutions.

  Polypeptides can be obtained from microorganisms isolated from nature (eg, contaminants, compost, water, etc.) using the probes described above, or DNA samples obtained directly from natural substances (eg, contaminants, compost, water, etc.). It can also be identified and obtained from other sources including. Techniques for directly isolating microorganisms and DNA from the natural environment are well known in the art. Polynucleotides encoding the polypeptides can then be obtained by similarly screening genomic DNA or cDNA libraries of other microorganisms or mixed DNA samples. Once the polynucleotide encoding the polypeptide is detected with a probe, the polynucleotide is isolated by utilizing techniques known to those skilled in the art (see, eg, Sambrook et al., 1989, supra). Or it can be cloned.

Polynucleotides The invention also relates to isolated polynucleotides that encode the polypeptides of the invention, as described herein.

  Techniques used for polynucleotide isolation or cloning are known in the art and include isolation from genomic DNA or cDNA, or combinations thereof. Cloning of polynucleotides from genomic DNA is performed, for example, by detecting cloned DNA fragments with shared structural mechanisms using the well-known polymerase chain reaction (PCR) or antibody screening of expression libraries. Is possible. For example, Innis et al. , 1990, PCR: A Guide to Methods and Applications, Academic Press, New York. Other nucleic acid amplification methods such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleotide based amplification (NASBA) can be used. The polynucleotide can be cloned from a Saccharothrix strain or related organism, and thus can be, for example, an allelic or species variant of a polypeptide encoding a region of the polynucleotide.

  Modification of a polynucleotide encoding a polypeptide of the invention may be necessary to synthesize a polypeptide that is substantially similar to the polypeptide. The term “substantially similar” to a polypeptide refers to a non-natural form of the polypeptide. These polypeptides may differ from the polypeptides isolated from natural sources in some manner, for example, variants that differ in specific activity, heat resistance, optimum pH, and the like. Variants can be constructed based on the polynucleotide represented as the mature polypeptide coding sequence of SEQ ID NO: 1 (eg, a subsequence thereof) and / or do not cause a change in the amino acid sequence of the polypeptide, It can be constructed by introducing nucleotide substitutions that correspond to the codon usage of the host organism intended for the production of the enzyme, or by introducing nucleotide substitutions that can result in different amino acid sequences. For an overview of nucleotide substitutions, see, eg, Ford et al. 1991, Protein Expression and Purification 2: 95-107.

Nucleic acid constructs The invention also relates to nucleic acid constructs comprising a polynucleotide of the invention operably linked to one or more control sequences that cause the coding sequence to be expressed in a suitable host cell under conditions compatible with the control sequences.

  A polynucleotide can be treated in a variety of ways to provide for expression of the polypeptide. Depending on the expression vector, treatment of the polynucleotide prior to insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

  The control sequence may be a promoter that is a polynucleotide recognized by the host cell for expression of the polynucleotide encoding the polypeptide of the invention. A promoter contains transcriptional control sequences that mediate expression of the polypeptide. A promoter can be any polynucleotide that exhibits transcriptional activity in host cells, including mutant, cleaved and hybrid promoters, as well as extracellular or intracellular polypeptides that are homologous or heterologous to the host cell. It can be obtained from the encoding gene.

  Examples of suitable promoters that direct transcription of the nucleic acid constructs of the present invention in bacterial host cells include Bacillus amyloliquefaciens α-amylase gene (amyQ), Bacillus licheniformis α-Bacillus licheniformis Gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus bacillus bacillus Bacteria (Bacillus subtil is) xylA and xylB genes, Bacillus thuringiensis cryIIIA gene (Agaisse and Lerecrus, 1994, Molecular Microbiology 13: 97-107), E. coli lac operoper, t. (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicolor agarase gene (dagA) and prokaryotic β-lactamase gene (Villa-Kamaoff et al., 1978, Proc. Natl. Acad.Sci.USA 75: 3727-3731), Are promoters derived from the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25). Additional promoters are described in “Useful proteins from recombinant bacteria”, Gilbert et al. , 1980, Scientific American 242: 74-94; and the aforementioned Sambrook et al. 1989. An example of a tandem promoter is disclosed in WO99 / 42835.

  Examples of suitable promoters that direct transcription of the nucleic acid constructs of the present invention in filamentous fungal host cells include the following: Aspergillus nidulans acetamidase, Aspergillus niger neutral α-amylase, Aspergillus Aspergillus niger acid stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae Protein Degrading enzyme, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787 pamphlet), Fusarium venenatum (Fusarium venenatum) 56900), Fusarium venenatum Daria (International Publication No. 00/56900 pamphlet), Fusarium venenatum Quinn (International Publication No. 00/56900 pamphlet), Rhisomkormi (Rhizomucor m ehei) lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei β-glucosidase, Trichoderma reesei derikorei serobiase Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei (Trichoderma reesei) reesei) endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesiei β, Trichoderma reesei xyderase i NA2-tpi promoter (a modified promoter derived from the Aspergillus neutral α-amylase gene, in which the untranslated leader is replaced with a non-translated leader derived from the Aspergillus triose phosphate isomerase gene; The non-translated leader is Aspergillus nidurans (Including a modified promoter from the Aspergillus niger neutral α-amylase gene substituted with an untranslated leader from the Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene). There are resulting promoters; and mutant, truncated and hybrid promoters thereof.

  Useful promoters in yeast hosts include Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae S cerevisiae S cerevisiae S Aldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae (UP) metallothione CUP It is obtained from the gene of Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al. 1992, Yeast 8: 423-488.

  The control sequence may also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator is operably linked to the 3'-end of the polynucleotide encoding the polypeptide. Any terminator that functions in the host cell can be used in the present invention.

  Preferred terminators for bacterial host cells are Bacillus clausii alkaline protease (aprH), Bacillus licheniformis α-amylase gene (amyL), and Escherichia coli ribosomal RNA from Escherichia coli B RNA. can get.

  Preferred terminators of filamentous fungal host cells are Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger α-Glua ligase α-glucose And from the gene for Fusarium oxysporum trypsin-like proteolytic enzyme.

  Preferred terminators for yeast host cells are the Saccharomyces cerevisiae enolase, the Saccharomyces cerevisiae gene cytochrome C (CYC1) and the Saccharomyces cerevisiae cerevisiae cerevisiae cerevisiae cerevisiae cerevisiae cerevisiae c Obtained from. Other useful terminators for yeast host cells are described in Romanos et al. , 1992.

  The control sequence may be an mRNA stabilization region downstream of the promoter and upstream of the coding sequence of the gene, which increases the expression of the gene.

  Examples of suitable mRNA stabilization regions are the Bacillus thuringiensis cryIIIA gene (WO 94/25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacter 17). : 3465-3471).

  The control sequence may also be a leader, ie, an untranslated region of the mRNA, which is important for translation by the host cell. The leader is operably linked to the 5'-end of the polynucleotide encoding the polypeptide. Any reader that is functional in the host cell may be used.

  Preferred leaders of filamentous fungal host cells are derived from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

  Preferred leaders for yeast host cells are Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae (Saccharomyces cerevisiae) It is derived from the gene of Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

  The control sequence is also a polyadenylation sequence that is operably linked to the 3′-end of the polynucleotide and, when transcribed, is a sequence that is recognized by the host cell as a signal to load polyadenosine residues into the transcribed mRNA. possible. Any polyadenylation sequence that functions in the host cell can be used.

  Preferred polyadenylation sequences for filamentous fungal host cells include Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger α-glucose It is derived from the gene of TAKA amylase and Fusarium oxysporum trypsin-like protease.

  Useful polyadenylation sequences for yeast host cells are described in Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

  The control sequence may also be a signal peptide coding region that codes for a signal peptide linked to the N-terminus of the polypeptide and directs the polypeptide into the cell's secretory pathway. The 5'-end of the polynucleotide coding sequence may inherently contain a signal peptide coding sequence originally linked to the translation reading frame, along with a segment of the coding sequence encoding the polypeptide. Alternatively, the 5'-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. If the coding sequence does not originally contain a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, the exogenous signal peptide coding sequence may simply replace the natural signal peptide coding sequence to enhance polypeptide secretion. However, any signal peptide coding sequence that sends the expressed polypeptide into the secretory pathway of the host cell may be used.

  Effective signal peptide coding sequences for bacterial host cells include Bacillus NCIB11837 maltogenic amylase, Bacillus licheniformis, subtilisin, Bacillus licheniformis β-lactamase, Bacillus licheniformis, Bacillus stearothermophilus α-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM) and a sequence derived from the gene of Bacillus subtilis prsA. Additional signal peptides are described in Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

  Effective signal peptide coding sequences for filamentous fungal host cells include Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKH amylase, insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei signal sequence derived from Rhizomucor miehei aspartic acid signal.

  Useful signal peptides for yeast host cells are derived from the genes for Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described in the aforementioned Romanos et al. , 1992.

  The control sequence may also be a propeptide coding sequence that encodes a propeptide position at the N-terminus of the polypeptide. The resulting polypeptide is known as a zymogen or propolypeptide (or zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. Propeptide coding sequences include Bacillus subtilis alkaline proteolytic enzyme (aprE), Bacillus subtilis neutral proteolytic enzyme (nprT), and Myceliophthola thermophila public 95 laccase. / 33836 pamphlet), Rhizomucor miehei aspartic proteinase and Saccharomyces cerevisiae α-factor gene.

  When both a signal peptide and a propeptide sequence are present, the propeptide sequence is located adjacent to the N-terminus of the polypeptide, and the signal peptide sequence is adjacent to the N-terminus of the propeptide sequence. Is located.

  It may be desirable to add regulatory sequences that regulate the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that turn gene expression on and off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or the GAL1 system can be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKAα-amylase promoter and the Aspergillus oryzae glucoamylase promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In the eukaryotic system, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate and the metallothionein gene that is amplified with heavy metals. In these instances, the polynucleotide encoding the polypeptide will be operably linked to a regulatory sequence.

Expression Vector The present invention also relates to a recombinant expression vector comprising the polynucleotide, promoter, and transcription and translation termination signals of the present invention. A recombinant expression vector in which various nucleotides and control sequences can be combined to contain one or more convenient restriction sites to allow insertion or substitution of a polynucleotide encoding a polypeptide at such sites. Is generated. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into a vector suitable for expression. In forming the expression vector, the coding sequence is located in the vector such that the coding sequence is operably linked with the appropriate control sequences for expression.

  The recombinant expression vector can be any vector (eg, a plasmid or virus) that can be conveniently subjected to recombinant DNA methods and can bring about the expression of the polynucleotide. The choice of vector will typically depend on the affinity of the vector for the host cell into which it is introduced. The vector can be a linear or closed circular plasmid.

  The vector can be a self-replicating vector (ie, a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication), eg, a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector may contain some means to ensure self-replication. Alternatively, the vector may be one that, when introduced into a host cell, is integrated into the genome and replicated together with the chromosomes integrated therein. Moreover, a single vector or plasmid, or two or more vectors or plasmids or transposons that contain together all the DNA introduced into the host cell genome may be used.

  The vector preferably contains one or more selectable markers that facilitate selection of transformed, transfected, transduced cells, etc. Selectable markers are genes whose products provide biocide or virus resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

  Examples of bacterial selection markers include Bacillus licheniformis or Bacillus subtilis dal gene or antibiotics such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance It is a marker that provides resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinotricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (Nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyl transferase) and trpC (anthranilate synthase), and their equivalents. Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes, and Streptomyces hygroscopicus spur cells (preferably used in the genus Streptomyces hygroscopicus spur cells)

  The vector preferably contains elements that allow integration of the vector into the genome of the host cell or autonomous replication of the vector in the cell independent of the genome.

  With respect to integration into the host cell genome, the vector may depend on the sequence of the polynucleotide encoding the polypeptide or any other element of the vector involved in integration into the genome by homologous or heterologous recombination. . Alternatively, the vector may contain additional polynucleotides that provide for integration by homologous recombination into the genome of the host cell at the exact location in the chromosome. In order to increase the likelihood of being integrated at the correct position, the integration element has a high degree of sequence identity to the corresponding target sequence to increase the probability of homologous recombination, 100-10,000 base pairs. A sufficient number of nucleic acids, such as 400 to 10,000 base pairs and 800 to 10,000 base pairs. The integration element can be any sequence that is homologous to the target sequence in the genome of the host cell. Furthermore, the integration element can be a non-coding or coding polynucleotide. On the other hand, the vector can be integrated into the genome of the host cell by non-homologous recombination.

  For autonomous replication, the vector may further include an origin of replication that allows the vector to replicate autonomously in the subject host cell. The origin of replication can be any plasmid replicator that mediates autonomous replication that functions in the cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that allows replication of a plasmid or vector in vivo.

  Examples of bacterial origins of replication include the origins of plasmids pBR322, pUC19, pACYC177 and pACYC184 that allow replication in E. coli, and pUB110, pE194, pTA1060 and pAMβ1 that allow replication in Bacillus. This is the origin of replication.

  Examples of origins of replication used in yeast host cells are 2 micron origins of replication, ARS1, ARS4, ARS1 and CEN3 combinations, and ARS4 and CEN6 combinations.

  Examples of useful origins of replication in filamentous fungal cells are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; 00/24883 pamphlet). Isolation of the AMA1 gene and construction of a plasmid or vector containing the AMA1 gene can be achieved according to the method disclosed in WO 00/24883.

  More than one copy of the polynucleotide of the invention may be inserted into the host cell to enhance production of the polypeptide. The number of copies of the polynucleotide can be increased by integrating at least one additional copy of the sequence into the host cell genome, or by including an amplifiable selectable marker gene in the polynucleotide, where Thus, cells containing an amplified copy of the selectable marker gene, and thus an additional copy of the polynucleotide, can be selected by culturing the cells in a suitable selectable agent.

  Techniques used to link the above elements to construct the recombinant expression vectors of the invention are well known to those skilled in the art (see, eg, Sambrook et al., 1989, supra).

Host cells The invention also relates to recombinant host cells comprising a polynucleotide of the invention operably linked to one or more control sequences that result in the production of a polypeptide of the invention. A construct or vector comprising the polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating redundant-chromosomal vector as described above. The term “host cell” encompasses any progeny of a parent cell that is not equivalent to the parent cell due to mutations that occur during replication. The choice of host cell will depend largely on the gene encoding the polypeptide and its source.

  A host cell can be any cell useful in the recombinant production of a polypeptide of the invention, eg, prokaryotic or eukaryotic.

  Prokaryotic host cells can be either gram positive or gram negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Examples include the genus Oceanobacillus, the genus Staphylococcus, the Streptococcus and the Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, and Iriobacter (Ilyobacter), Neisseria, Pseudomonas, Salmonella and Ureaplasma.

  Bacterial host cells include, but are not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus cils, Bacillus clausii, Bacillus coagulans, Bacillus films, Bacillus laurus, Bacillus lentils rmis), Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus subtilis cells. It can be any Bacillus cell.

  Bacterial host cells also include, but are not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus sp., And Streptococcus sp. It can be any Streptococcus cell, including any Streptococcus cell.

  Bacterial host cells may also include, but are not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coliploidum, Streptomyces streptomyces. griseus), and any Streptomyces cell, including Streptomyces lividans cells.

  Introduction of DNA into Bacillus cells can be accomplished by protoplast transformation (see, eg, Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), competent cell transformation (eg, Young and Spizzen, 1961, J. Bacteriol. 81: 823-829, or Dubau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation (eg, Shigekawa and Dower , 1988, Biotechniques 6: 742-751) or by conjugation (eg, Koehler and Thorne, 1987, J. Bacteri. ol.169: 5271-5278). Introduction of DNA into E. coli cells can be accomplished by protoplast transformation (see, eg, Hanahan, 1983, J. Mol. Biol. 166: 557-580) or by electroporation (eg, Dower. et al., 1988, Nucleic Acids Res. 16: 6127-6145). Introduction of DNA into Streptomyces cells can be accomplished by protoplast transformation electroporation (see, eg, Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), conjugation ( See, eg, Mazodier et al., 1989, J. Bacteriol.171: 3583-3585) or by transduction (eg, Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). Introduction of DNA into Pseudomonas cells can be accomplished by electroporation (see, for example, Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or by conjugation (eg, Pinedo). and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Introduction of DNA into Streptococcus cells can be accomplished by natural competence (see, for example, Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), protoplast transformation (see, for example, Catt and Jollick, 1991). , Microbios 68: 189-207), electroporation (see, for example, Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or by joining (eg, Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any of the methods known in the art for introducing DNA into host cells can be used.

  The host cell can also be a eukaryotic organism such as a mammalian, insect, plant or fungal cell.

  The host cell may be a fungal cell. As used herein, “fungi” refers to Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota, and Omycota and all nutrients Includes sporulating fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, as defined by Cambridge).

  The fungal host cell can be a yeast cell. As used herein, “yeast fungus” belongs to ascomycetous yeast (Endomycetals), basidiomycetous yeast, and yeast belongs to imperfect fungi (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of the present invention, yeast is selected from Biology and Activities of Yeast (Skinner, Passmore and Davenport, Soc. App. Bacteriol. Symposium 9 Symposium. 1980).

  Yeast host cell, Kluyveromyces lactis (Kluyveromyces lactis), Saccharomyces Seth callus Bergen cis (Saccharomyces carlsbergensis), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces Jiasutachikusu (Saccharomyces diastaticus), Saccharomyces Dougurashii (Saccharomyces douglasii), Saccharomyces Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces obiformis (Saccharomyces ovifumis) ormis or Yarrowia lipolytica cells such as Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Saccharomyces (Schizosaccharomyces) or Yarrowia cells.

  The fungal host cell can be a filamentous fungal cell. “Filamentous fungi” include all filament forms of the fungal phylum (Emycota) and Omycota genus (as defined in Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan and other complex polysaccharides. Vegetative growth is by hyphal extension and carbon catabolism is absolutely aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is due to budding of unicellular fronds and carbon catabolism can be fermentative.

  Filamentous fungal host cells include Acremonium, Aspergillus, Aureobasidium, Bjerkandara, Celiporiopsis, Chrysosporis Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnacorth, Magnacorth, Magnaportor Myceliophthora, neo-kali mastic Genus (Neocallimastix), Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phleour, Phlomyur, Phlomyur (Schizophyllum), Talaromyces, Thermoascus, Thielavia, Tolypocladum, Trametes or Trachoderma cells, or Trichoderma derma.

  For example, filamentous fungal host cells include Aspergillus awagi, Aspergillus fosidus, Aspergillus fumigatus, Aspergillus fumigatus, Aspergillus fumigatus (Aspergillus fumigatus) Niger (Aspergillus niger), Aspergillus oryzae, Yakerotake (Bjerkandara adusta), Seripoliopsis aneirina, Seriporiopsis Carigiairis (Ceripopsis carregia), Seripoliopsis gilvessens (Ceriporiopsis girubescens), Seripolyopsis pannosita (Ceriporiopsis pannocinta), Seripoliopis cerevisiae (Ps) Suborimispora (Ceriporiopsis subvermisspora), Chrysosporium inops, Chrysosporium keratinofilm, Chrysosporium keratinophilum Chrysosporium lumpnowense, Chrysosporium merdarium, Chrysosporium penicola, Chrysosporium penicola, Chrysosporium crispium Ssporium zonatum, Coprinus cinereus, Criolus hirsutus, Fusarium bacterioides ), Fusarium cerrealis, Fusarium crouchwellense, Fusarium culumum, Fusarium gramumum, Fusarium gramumumum, Fusarium gramumumum ), Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium roseum, Fusarium roseum Fusarium sulbuumum, Fusarium sarcochrome, Fusarium sulphulium, Fusarium sulphumum, Fusarium sulphumum, Fusarium sulphumum. trichothecioids, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor mihei ei), Myceliophthora thermophila (Myceliophthora thermophila), Neurospora crassa (Neurospora crassa), Penicillium Purupurogenumu (Penicillium purpurogenum), Fanerokete chrysosporium (Phanerochaete chrysosporium), Furebia-radiata (Phlebia radiata), Pureurotsusu-Eringii ( Pleurotus eryngii, Thielavia terrestris, Trametes vilosa, Trametes versicolor, Trichoderma harzianum Trichoderma harzianum), Trichoderma Koningii (Trichoderma koningii), Trichoderma longibrachiatum (Trichoderma longibrachiatum), it may be a Trichoderma reesei (Trichoderma reesei) or Trichoderma viride (Trichoderma viride) cells.

  Fungal cells can be transformed by processes including protoplast formation, protoplast transformation, and cell wall regeneration in a manner known per se. Suitable techniques for transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al. , 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474, and Christensen et al. , 1988, Bio / Technology 6: 1419-1422. Suitable methods for transformation of Fusarium species are described in Malardier et al. 1989, Gene 78: 147-156 and WO 96/00787. Yeast has been described by Becker and Guarente, In Abelson, J. MoI. N. and Simon, M .; I. , Editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc. , New York; Ito et al. , 1983, J. et al. Bacteriol. 153: 163; and Hinnen et al. 1978, Proc. Natl. Acad. Sci. USA 75: 1920 can be used for transformation.

Methods of Production The invention also includes: (a) culturing cells that produce the polypeptide in wild-type form under conditions that permit production of the polypeptide; and (b) recovering the polypeptide. It relates to a method for producing a polypeptide of the invention. In a preferred embodiment, the cell is a Saccharothrix cell. In a more preferred embodiment, the cell is a Saccharothrix australiansis cell.

  The invention also includes: (a) culturing a recombinant host cell of the invention under conditions that permit production of the polypeptide; and (b) recovering the polypeptide. It relates to a method of generating.

  Host cells are cultured in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cells are cultured in a suitable medium and under conditions that permit the expression and / or isolation of the polypeptide, by shake flask culture, and in small or large scale fermentations in laboratory or industrial fermenters. (Including continuous, batch, fed-batch or solid state fermentation). Culturing is performed using techniques known in the art in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts. Suitable media are available from commercial suppliers or can be prepared according to published compositions (eg, catalog of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from the cell lysate.

  The polypeptide can be detected using methods known in the art specific for the polypeptide. These detection methods include, but are not limited to, the use of specific antibodies, formation of enzyme products, or disappearance of enzyme substrates. For example, enzymatic activity may be determined using an enzyme assay.

  The polypeptide can be recovered using methods known in the art. For example, the polypeptide can be recovered from the nutrient medium by conventional techniques including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation or precipitation.

  Polypeptides are not particularly limited to obtain substantially pure polypeptides, but include chromatography (eg, ion exchange, affinity, hydrophobicity, chromatofocusing and size exclusion), electrophoretic techniques (eg, Preparative isoelectric focusing), differential solubility (eg, ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, Jans and Ryden, VCH Publishers, New York, 1989). It can be purified by various techniques known in the art.

  In an alternative embodiment, the polypeptide is not recovered and a host cell of the invention that expresses the polypeptide can be used as the source of the polypeptide.

Plants The invention also provides an isolated plant, eg, a transgenic plant, plant part, or plant, that contains a polynucleotide of the invention so that the polypeptide or domain can be expressed and produced in recoverable amounts. Also related to cells. The polypeptide or domain may be recovered from the plant or plant part. Alternatively, a plant or plant part containing a polypeptide or domain may be used as it is for the purpose of improving the quality of food or feed, for example, improving nutritional value, palatability and fluidity, or destroying non-nutritive factors. .

  The transgenic plant may be a dicotyledonous plant (dicot) or a monocotyledonous plant (monocot). Examples of monocotyledonous plants include grasses such as forage grass (Nagahagusa, Strawberry jumpweed (Poa)), bovine rush (Festuca), ryegrass (Lolium), cold grasses such as Nukabo (Agrostis), and cereals, for example , Wheat, oats, rye, barley, rice, sorghum and corn (corn).

  Examples of dicotyledonous plants include legumes such as tobacco and waxy beans, potatoes, sugar beets, peas, kidney beans and soybeans, and cruciferous plants such as cauliflower and rapeseed (Brassicaceae) and related model organisms. There is an Arabidopsis thaliana.

  Examples of plant parts include stems, callus, leaves, roots, fruits, seeds and tubers and individual tissues containing these parts, such as epidermis, gingiva, soft tissue, duct tissue, meristems, and the like. Certain plant cell compartments such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and cytoplasm are also considered plant parts. Furthermore, any plant cell is considered to be a plant part, regardless of which tissue it originates from. Similarly, plant parts such as certain tissues and cells isolated to facilitate use of the present invention are also considered plant parts, such as embryos, endosperm, aleurone and seed coats.

  Such plants, plant parts, and plant cell progeny are also within the scope of the present invention.

  Transgenic plants or plant cells that express a polypeptide or domain can be constructed according to methods known in the art. Briefly, after incorporating one or more expression constructs encoding a polypeptide or domain into a plant host cell genome or chloroplast genome, the resulting modified plant or plant cell is transformed into a transgenic plant or plant cell. Build by growing.

  The expression construct is preferably a nucleic acid construct comprising a polynucleotide encoding a polypeptide or domain operably linked to suitable regulatory sequences necessary for expression of the polynucleotide in a selected plant or plant part. Furthermore, the expression construct may include a selection marker useful for identifying a plant cell in which the expression construct is incorporated, and a DNA sequence necessary for introduction of the construct into the target plant (the latter is a DNA introduction method to be used). Depending on).

  The choice of promoter and terminator sequences, and optionally regulatory sequences such as signal or transit sequences, is determined, for example, depending on when, where and how the polypeptide or domain is desired to be expressed. For example, the expression of a gene encoding a polypeptide or domain can be constitutive or inducible, or it can be developmental, stage or tissue specific, and the gene product can be expressed in specific species such as seeds or leaves. It may be targeted to a tissue or plant part. Regulatory sequences are described, for example, in Tague et al. , 1988, Plant Physiology 86: 506.

  For constitutive expression, the 35S-CaMV, maize ubiquitin 1 or rice actin 1 promoter may be used (Franck et al., 1980, Cell 21 285-294; Christensen et al., 1992, Plant Mol. Biol. 18). : 675-689; Zhang et al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be, for example, storage sink tissues such as seeds, potato tubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24: 275- 303), or a promoter derived from metabolic sink tissue such as meristem (Ito et al., 1994, Plant Mol. Biol. 24: 863-878), seed-specific such as rice-derived glutelin, prolamin, globulin, or alvin promoter Promoters (Wu et al., 1998, Plant Cell Physiol. 39: 885-889), Vicia faba promoter from legumin B4, and Vicia (Vicia) Faba) unknown seed protein gene (Conrad et al., 1998, J. Plant Cell Physiol. 152: 708-711), oil seed body protein derived promoter (Chen et al., 1998, Plant Physiol. 39). : 935-941), a storage protein napA promoter from Brassica napus, or any other seed-specific promoter known in the art, for example as described in WO 91/14772. Further, the promoter may be the following: a leaf-specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol. 102: 991-1000), the chlorella virus adenine methyltransferase gene promoter ( Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), rice-derived aldP gene promoter (Kagaya et al., 1995, Mol. Gen. Genet. 248: 668-674), or potato pin2 promoter Injury inducible promoters (Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Similarly, promoters may be induced by abiotic treatments such as temperature, drying, or changes in salinity, or substances added extracellularly that activate the promoter, such as ethanol, estrogen, and ethylene. It may be derived from plant hormones (such as abscisic acid and gibberellic acid) and heavy metals.

  Promoter enhancer elements may be used to achieve higher expression of a polypeptide or domain in plants. For example, the promoter enhancer element may be an intron, which is placed between the promoter and a polynucleotide encoding a polypeptide or domain. For example, Xu et al., 1993 (supra), disclosed the use of the first intron of rice actin 1 for the purpose of enhancing expression.

  The selection marker gene and any other part of the expression construct may be selected from those available in the art.

  Nucleic acid constructs are prepared by conventional techniques known in the art (Gasser et al., 1990, Science 244) such as Agrobacterium-mediated transformation, virus-mediated transformation, microinjection, particle gun, particle gun, and electroporation. : 1293; Potrykus, 1990, Bio / Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

  Agrobacterium tumefaciens-mediated gene transfer is a method for producing transgenic dicotyledonous plants (for an overview, see Hoykas and Schiperoort, 1992, Plant Mol. Biol. 19: 15-38) and single Although it is a method of transforming cotyledons, other transformation methods may be used for these plants. Methods for producing transgenic monocotyledons are embryogenic callus or developing embryo particle guns (fine gold or tungsten particles coated with transforming DNA) (Christou, 1992, Plant J. 2: 275-). 281; Shimamoto, 1994, Curr. Opin.Biotechnol.5: 158-162; Vasil et al., 1992, Bio / Technology 10: 667-674). Another method for transformation of monocotyledons is described by Omiruleh et al. 1993, Plant Mol. Biol. 21: 415-428, based on protoplast transformation. Alternative transformation methods are described in US Pat. Nos. 6,395,966 and 7,151,204, both of which are hereby incorporated by reference in their entirety. There is something.

  After transformation, transformants incorporating the expression construct are selected and regenerated into whole plants by methods known in the art. In many cases, transformation methods are designed for selective elimination of selection genes, either during regeneration or at the time of subsequent production, but this is, for example, by two separate T-DNA constructs. Co-transformation or site-specific excision of selected genes with specific recombinases is used.

  In addition to direct transformation of a specific plant genotype containing the construct of the present invention, a transgenic plant can also be produced by crossing a second plant not containing the construct with a plant containing the construct. For example, a construct encoding a polypeptide or domain can be introduced into a particular plant variant by crossing, without the need to directly transform the plant of that particular variant. Accordingly, the present invention includes not only plants regenerated directly from cells transformed according to the present invention, but also the progeny of such plants. As used herein, “progeny” may refer to progeny of any generation of a parent plant prepared according to the present invention. Such progeny may comprise a DNA construct prepared according to the present invention. As a result of crossing, the transgene is introduced into the plant system by cross-pollination of the starting system and the donor plant system. A non-limiting example of such a step is described in US Pat. No. 7,151,204.

  Plants can also be produced by backcrossing. For example, plants include plants referred to as backcross genotypes, lines, inbreds, or hybrids.

  Genetic markers may be used to facilitate introgression of one or more of the invention's trass genes from one genetic background to another. Marker-based selection offers the advantage over conventional breeding that it can be used to avoid errors caused by phenotypic changes. In addition, genetic markers can provide data regarding the relative proportion of elite germplasm in individual offspring of a particular cross. For example, when a plant with the desired characteristics, usually with a non-agriculturally desirable genetic background, is crossed with an elite parent, not only has the desired characteristics using genetic markers, but also the desired reproduction at a relatively large rate. Offspring including quality can be selected. In this way, the number of generations required to transfer one or more features to a particular genetic background is minimized.

  The invention also includes (a) culturing a transgenic plant or plant cell comprising a polynucleotide encoding the polypeptide or domain under conditions that permit production of the polypeptide or domain; and (b) the polypeptide or It also relates to a method for producing a polypeptide or domain of the invention comprising the step of recovering the domain.

Signal Peptides and Propeptides The present invention further relates to an isolated polynucleotide encoding a signal peptide comprising or consisting of amino acids 1-29 of SEQ ID NO: 2. The present invention also relates to an isolated polynucleotide encoding a propeptide comprising or consisting of amino acids 30-188 of SEQ ID NO: 2. The present invention also relates to an isolated polynucleotide encoding a signal peptide and propeptide comprising or consisting of amino acids 1-188 of SEQ ID NO: 2. The polynucleotide may further comprise a gene encoding a protein operably linked to the signal peptide and / or propeptide. The protein is preferably foreign to the signal peptide and / or propeptide. In one aspect, the polynucleotide encoding the signal peptide is nucleotides 101-187 of SEQ ID NO: 1. In another aspect, the polynucleotide encoding the propeptide is nucleotides 188-664 of SEQ ID NO: 1. In another aspect, the polynucleotide encoding the signal peptide and propeptide is nucleotides 101-664 of SEQ ID NO: 1.

  The invention also relates to nucleic acid constructs, expression vectors and recombinant host cells comprising such polynucleotides.

Detergent Composition In one embodiment, the present invention relates to a detergent composition comprising an enzyme of the present invention together with one or more additional cleaning composition ingredients. Accordingly, in one embodiment, the invention provides at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66% relative to the mature polypeptide of SEQ ID NO: 2. At least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91% , At least 9 %, At least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity and having protease activity The present invention relates to a detergent composition comprising a polypeptide.

  Selection of additional ingredients is within the skill of the artisan and includes conventional formulation ingredients including the exemplary non-limiting ingredients described below. Ingredient selection may include consideration of the type of fabric to be cleaned, the type and / or extent of contaminants, the temperature at which cleaning is performed, and the formulation of the detergent product with regard to fabric handling. The components described below are categorized by a general header that follows a particular functional group, but this should not be construed as limiting and additional functional groups will be present in the component as will be appreciated by those skilled in the art. It may be included.

Enzyme of the Invention In one embodiment of the invention, the polypeptide of the invention comprises 0.001 to 200 mg protein, for example 0.005 to 100 mg protein, preferably 0.01 to 50 mg, per liter of washing solution. Protein may be added to the detergent composition in an amount corresponding to 0.05-20 mg protein, more preferably 0.1-10 mg protein.

  The enzyme of the detergent composition of the present invention is, for example, a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative such as an aromatic boric acid ester, or 4-formylphenylboron. Conventional stabilizers such as phenylboronic acid derivatives such as acids may be used and the compositions are described, for example, in WO 92/19709 and WO 92/19708. May be prepared as described. Alternatively, the variants of the invention may be stabilized using peptide aldehydes or ketones such as those described in WO 2005/105826 and WO 2009/118375.

  The polypeptides of the present invention may also be incorporated into the detergent formulations disclosed in WO 97/07202, which is incorporated herein by reference.

Surfactant The detergent composition comprises one or more surfactants that can be anionic and / or cationic and / or nonionic and / or semipolar and / or amphoteric or mixtures thereof. May be. In certain embodiments, the detergent composition comprises a mixture of one or more nonionic surfactants and one or more anionic surfactants. The surfactant is typically present at a level of about 0.1% to about 60%, such as about 1% to about 40% or about 3% to about 20% or about 3% to about 10%. The surfactant is selected based on the desired cleaning application and includes any conventional surfactant known in the art. Any laundry surfactant known in the art that can be used in laundry detergents can be utilized.

  When included, the detergent is typically about 1% to about 40% by weight anionic surfactant, such as about 5% to about 15% or about 5% to about 30%, including about 20% to about 25%. Will contain. Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular linear alkyl benzene sulfonates (LAS), isomers of LAS, branched alkyl benzene sulfonates (BABS), phenylalkane sulfonates, α-olefins. Sulfonate (AOS), olefin sulfonate, alkene sulfonate, alkane-2,3-diylbis (sulfate), hydroxyalkane sulfonate and disulfonate, alkyl sulfate (AS) such as sodium dodecyl sulfate (SDS), aliphatic alcohol sulfate (FAS), primary alcohol sulfate (PAS), alcohol ether sulfate (Also known as alcohol ethoxy sulfate or aliphatic alcohol ether sulfate) Α-sulfo fatty acid methyl ester (α-SFMe or SES), including AEOS or FES), secondary alkane sulfonate (SAS), paraffin sulfonate (PS), ester sulfonate, sulfonated fatty acid glycerol ester, methyl ester sulfonate (MES) ), Alkyl- or alkenyl succinic acid, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, sulfosuccinic acid or soap diesters and monoesters, and combinations thereof.

  When included, the detergent typically comprises from about 1% to about 40% by weight of a cationic surfactant. Non-limiting examples of cationic surfactants include alkyl dimethyl ethanol quaternary amine (ADMEAQ), cetyl trimethyl ammonium bromide (CTAB), dimethyl distearyl ammonium chloride (DSDMAC), alkyl benzyl dimethyl ammonium, alkyl tert. Examples include quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA), and combinations thereof.

  When included, the detergent is typically about 3% to about 5% or about 8% to about 12%, such as about 0.5% to about 30%, especially about 1% to about 20%, about 3%. From about 0.2% to about 40% by weight of nonionic surfactant, such as up to about 10%. Non-limiting examples of nonionic surfactants include alcohol ethoxylate (AE or AEO), alcohol propoxylate, propoxylated fatty alcohol (PFA), ethoxylated and / or propoxylated fatty acid alkyl esters, etc. Alkoxylated fatty acid alkyl ester, alkylphenol ethoxylate (APE), nonylphenol ethoxylate (NPE), alkyl polyglycoside (APG), alkoxylated amine, fatty acid monoethanolamide (FAM), fatty acid diethanolamide (FADA), ethoxylated fatty acid mono Ethanolamide (EFAM), propoxylated fatty acid monoethanolamide (PFAM), polyhydroxyalkyl fatty acid amide, or N-acyl N-al of glucosamine Le derivative (glucosamide GA or fatty acid glucamides FAGA), as well as products available under the trade name SPAN, and TWEEN, as well as combinations thereof.

  When included, the detergent will usually contain from about 1% to about 40% by weight of a semipolar surfactant. Non-limiting examples of semipolar surfactants include alkyldimethylamine oxide, N- (cocoalkyl) -N, N-dimethylamine oxide and N- (tallow-alkyl) -N, N-bis (2- Amine oxides (AO) such as hydroxyethyl) amine oxide, fatty acid alkanolamides and ethoxylated fatty acid alkanolamides, and combinations thereof.

  When included, the detergent will usually contain from about 1% to about 40% by weight of zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaine, alkyl dimethyl betaine and sulfobetaine, and combinations thereof.

Hydrotropes Hydrotropes are compounds that solubilize hydrophobic compounds in aqueous solutions (or, conversely, polar substances in a nonpolar environment). Typically, hydrotropes have both hydrophilic and hydrophobic properties (so-called amphiphilic properties known from surfactants); however, the molecular structure of hydrotropes is generally self-sufficient. See, for example, the review by Hodgdon and Kaler (2007), Current Opinion in Colloid & Interface Science 12: 121-128. Hydrotropes do not exhibit the limiting concentration that causes self-aggregation above which is found for surfactants and lipids that form micelle phases, lamellar phases or other well-defined mesophases. Instead, many hydrotropes exhibit a continuous type of agglomeration process where the agglomerate size increases with increasing concentration. However, many hydrotropes modify the phase behavior, stability and colloidal properties of systems containing substances of polar and non-polar character, including mixtures of water, fats, surfactants and polymers. Hydrotropes are traditionally used in industry from pharmaceutical, personal care, food to technical applications. By using hydrotropes in the detergent composition, for example, more concentrated formulations of surfactants (liquid detergents by eliminating water) without inducing undesirable phenomena such as phase separation or high viscosity Can be made as compact as possible).

  The detergent may contain 0-5% by weight hydrotrope, such as about 0.5 to about 5% or about 3% to about 5%. Any hydrotrope known in the art used in detergents can be utilized. Non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymenesulfonate, amine oxide, Examples include alcohols and polyglycol ethers, sodium hydroxynaphthalene, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, and combinations thereof.

Builders and Cobuilders Detergent compositions may contain from about 0 to 65% by weight of detergent builder or cobuilder, such as from about 5% to about 50%, or mixtures thereof. In dishwashing detergents, the level of builders is typically 40-65%, especially 50-65%. Builders and / or co-builders can in particular be chelating agents that form water-soluble complexes with Ca and Mg. Any builder and / or cobuilder known in the art for use in laundry detergents can be utilized. Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, sodium metasilicate, etc. Soluble silicates, layered silicates (eg, SKS-6 from Hoechst), 2-aminoethane-1-ol (MEA), diethanolamine (DEA, also known as iminodiethanol), triethanolamine (TEA, Also included are ethanolamines such as 2,2 ′, 2 ″ -nitrilotriethanol), carboxymethylinulin (CMI), and combinations thereof.

  The detergent composition may also contain 0 to 65% by weight of detergent cobuilder, such as about 5% to about 40%, or mixtures thereof. The detergent composition may contain cobuilder alone or in combination with a builder such as a zeolite builder. Non-limiting examples of cobuilders include polyacrylate homopolymers such as poly (acrylic acid) (PAA) or copoly (acrylic acid / maleic acid) (PAA / PMA) or copolymers thereof. Further non-limiting examples include chelating agents such as citrates, aminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl- or alkenyl succinic acids. Further specific examples include 2,2 ′, 2 ″ -nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N, N ′. -Disuccinic acid (EDDS), methylglycine diacetic acid (MGDA), glutamic acid-N, N-diacetic acid (GLDA), ethylenediaminetetra (methylenephosphonic acid) (HEDP), ethylenediaminetetra (methylenephosphonic acid) (EDTMPA), diethylenetriamine Epentakis (methylenephosphonic acid) (DTPMPA), N- (2-hydroxyethyl) iminodiacetic acid (EDG), aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N, N-diacetic acid (ASDA) Aspartic acid-N-monopropionic acid ASMP), iminodisuccinic acid (IDA), N- (2-sulfomethyl) aspartic acid (SMAS), N- (2-sulfoethyl) aspartic acid (SEAS), N- (2-sulfomethyl) glutamic acid (SMGL), N- ( 2-sulfoethyl) glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), α-alanine-N, N-diacetic acid (α-ALDA), serine-N, N-diacetic acid (SEDA), isoserine-N, N-diacetic acid (ISDA), phenylalanine-N, N-diacetic acid (PHDA), anthranilic acid-N, N-diacetic acid (ANDA), sulfanilic acid-N, N-diacetic acid (SLDA), taurine-N, N-diacetic acid (TUDA) and sulfomethyl-N, N-diacetic acid (SMDA), N- (2-hydroxyethyl) -ethylidene diamine -N, N ', N'-triacetic acid (HEDTA), diethanolglycine (DEG), diethylenetriaminepenta (methylenephosphonic acid) (DTPMP), aminotris (methylenephosphonic acid) (ATMP), and combinations and salts thereof Is mentioned. Examples of further builders and / or co-builders are disclosed, for example, in WO 09/102854, US Pat. No. 5,977,053.

Bleaching system The detergent may contain 0 to 10 weights of bleaching system, such as about 1% to about 5%. Bleaching systems known in any technical field used in laundry detergents can be utilized. Suitable bleaching system components include bleach catalysts, photobleaching, bleach activators, sources of hydrogen peroxide such as sodium percarbonate and sodium perborate, preformed peracids, and mixtures thereof. It is done. Suitable pre-formed peracids include, but are not limited to, peroxycarboxylic acids and salts, percarbonate and salts, perimidic acid and salts, peroxymonosulfuric acid and salts such as oxone (R), and these Of the mixture. Non-limiting examples of bleaching systems include, for example, alkali metals such as perboric acid (usually monohydrate or tetrahydrate), percarbonate, persulfuric acid, perphosphoric acid, sodium salt of persilicic acid. Peroxide-based bleaching systems that can include inorganic salts, including salts, in combination with a peracid-forming bleach activator. As used herein, a bleach activator means a compound that reacts with a peroxygen bleach-like hydrogen peroxide to form a peracid. The peracid formed in this way constitutes an activated bleach. Suitable bleach activators for use herein include those belonging to the esteramide, imide or anhydride class. Preferred examples include tetraacetylethylenediamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl) oxy] benzenesulfonate (ISONOBS), diperoxide decanoic acid, 4- (dodecanoyloxy) benzenesulfonate ( LOBS), 4- (decanoyloxy) benzenesulfonate, 4- (decanoyloxy) benzoate (DOBS), 4- (decanoyloxy) benzoate (DOBS), 4- (nonanoyloxy) benzenesulfonate (NOBS), And / or those disclosed in WO98 / 17767. A specific family of subject bleach activators is disclosed in EP 624154, with acetyl triethyl citrate (ATC) being a particularly preferred family. ATC or short chain triglyceride-like triacins have the advantage of being environmentally friendly because they are ultimately broken down into citric acid and alcohol. Furthermore, acetyl triethyl citrate and triacetin have good hydrolytic stability in the product upon storage, which is an effective bleach activator. Finally, ATC provides a good supplement to laundry additives. Alternatively, the bleaching system can comprise, for example, an amide, imide or sulfone type peroxide. The bleaching system may also include a peracid such as 6- (phthalimido) peroxyhexanoic acid (PAP). The bleaching system may also contain a bleach catalyst. In some embodiments, the bleach component is an organic catalyst having the formula:
(Iii) and mixtures thereof wherein each R ¹ is independently a branched alkyl group containing 9 to 24 carbons or a linear alkyl group containing 11 to 24 carbons, preferably , each R ¹ is independently a linear alkyl group containing 9 to 18 or a branched alkyl group or a 11 to 18 carbons containing carbon, each R ¹ and more preferably, independently 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl and Selected from the group consisting of iso-pentadecyl). Other exemplary bleaching systems are described, for example, in International Publication No. 2007/087258, International Publication No. 2007/087244, International Publication No. 2007/087259, International Publication No. 2007/088724. Yes. A suitable photobleaching can be, for example, a sulfonated aene phthalocyanine.

Polymer detergents contain 0-10% by weight of polymer, such as 0.5-5%, 2-5%, 0.5-2% or 0.2-1%. Any polymer known in the art used in detergents can be utilized. The polymer can function as a co-builder as described above, or can provide anti-staining, fiber protection, contaminant release, migration prevention, oil cleaning and / or antifoam properties. Some polymers may have more than one of the above properties and / or more than one of the following motifs. Exemplary polymers include (carboxymethyl) cellulose (CMC), poly (vinyl alcohol) (PVA), poly (vinyl pyrrolidone) (PVP), poly (ethylene glycol) or poly (ethylene oxide) (PEG), ethoxylated Poly (ethyleneimine), carboxymethylinulin (CMI), and polycarboxylates such as PAA, PAA / PMA, poly-aspartic acid, and lauryl methacrylate / acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) and Silicone, copolymer of terephthalic acid and oligomeric glycol, copolymer of poly (ethylene terephthalate) and poly (oxyethene terephthalate) (PET-POET), PVP, poly (vinylimidazole) (PVI), poly (Vinylpyridine -N- oxide) (PVPO or PVPNO), as well as polyvinyl pyrrolidone - vinyl imidazole (PVPVI) and the like. Further exemplary polymers include sulfonated polycarboxylates, polyethylene oxide and polypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Other exemplary polymers are disclosed, for example, in WO 2006/130575. Also contemplated are salts of the above polymers.

Fabric Tone The detergent composition of the present invention may also include a fabric toning, such as a dye or pigment, which when incorporated into a detergent composition, the fabric contains the detergent composition. It is possible to change the color of the fabric through absorption / reflection of visible light when it is brought into contact with the fabric. The fluorescent whitening agent emits at least some visible light. In contrast, fabric toning agents change the surface shade by absorbing at least a portion of the visible light spectrum. Suitable fabric toning agents include dyes and dye-clay composites, and may also include pigments. Suitable dyes include micromolecular dyes and polymeric dyes. Suitable micromolecular dyes include, for example, WO 2005/03274 pamphlet, WO 2005/03275 pamphlet, WO 2005/03276 pamphlet and European Patent No. 1876226 (refer to the present specification by reference). Dye Index of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, or a mixture thereof described in US Pat. ) Micromolecular dyes selected from the group consisting of dyes belonging to the classification. The detergent composition comprises about 0.00003% to about 0.2%, about 0.00008% to about 0.05% or even about 0.0001% to about 0.04% by weight of fabric. It is preferable that a colorant is included. The composition may comprise from 0.0001% to 0.2% by weight of a fabric toning, which may be particularly preferred when the composition is in the form of a unit dose pouch. Suitable toning agents are also disclosed, for example, in WO 2007/087257, WO 2007/087243.

(Additional) enzymes Detergent additives and detergent compositions are for example proteolytic enzymes, lipases, cutinases, amylases, carbohydrases, cellulases, pectinases, mannanases, arabinases, galactanases, xylanases, oxidases, such as laccases and / or peroxidases One or more [additional] enzymes, such as

  In general, the properties of the selected enzyme should be compatible with the selected detergent (ie, optimal pH, affinity with other enzymatic and non-enzymatic formulation ingredients, etc.) and the enzyme is in an effective amount. Should exist.

  Cellulases: Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein modified mutants are included. Suitable cellulases include, for example, US Pat. No. 4,435,307, US Pat. No. 5,648,263, US Pat. No. 5,691,178, US Pat. No. 5,776. , 757 and WO 89/09259, produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum fungi. Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia avia), include Acremonium (Acremonium) derived cellulase.

  Particularly preferred cellulases are alkaline or neutral cellulases that have benefits with respect to color handling. Examples of such cellulases are EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. It is a cellulase described in a pamphlet. Other examples are WO 94/07998, EP 0 531 315, US Pat. No. 5,457,046, US Pat. No. 5,686,593, US Pat. Cellulase variants such as those described in US Pat. No. 5,763,254, WO 95/24471, WO 98/12307, and PCT / DK98 / 00299. It is.

  Commercially available cellulases include Celluzyme ™ and Carezyme ™ (Novozymes A / S), Clazinase ™ and Puradax HA ™ (Genencor International Inc.) and KAC-500 (B) ™. (Kao Corporation).

  Proteases: Suitable proteases include those of animal, vegetable or microbial origin. Those derived from microbial properties are preferred. Chemically modified or protein modified mutants are included. The proteolytic enzyme may be a serine proteolytic enzyme or a metalloprotease, preferably an alkaline microbial proteolytic enzyme or a trypsin-like proteolytic enzyme. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279) ). Examples of trypsin-like proteases are trypsin (eg from porcine or bovine) and Fusarium proteolytic enzymes as described in WO 89/06270 and WO 94/25583.

  Examples of useful proteases are the variants described in WO 92/19729, WO 98/2015, WO 98/2016, and WO 98/34946. In particular, variants with substitutions at one or more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274.

  Preferred commercially available proteolytic enzymes include Alcalase (TM), Savinase (TM), Primease (TM), Duralase (TM), Esperase (TM) and Kannase (TM) (Novozymes A / S), Maxatase (TM) ), Maxcal (TM), Maxapem (TM), Properase (TM), Purefect (TM), Perfect OxP (TM), FN2 (TM) and FN3 (TM) (Genencor International Inc.).

  Lipases and cutinases: Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein modified mutants are included. Examples include those derived from Thermomyces as described in European Patent No. 258 068 and European Patent No. 305 216, e.g. Lipases from T. lanuginosus (previously named Humicola lanuginosa); described in WO 96/13580, for example H. Cutinase from Humicola, such as H. insolens; Alkali (P. alcali) gene or P. P. pseudoalcali gene (European Patent No. 218 272), P. p. P. cepacia (EP 331 376), P. cepacia. P. stutzeri (UK Patent No. 1,372,034), P. stutzeri. P. fluorescens, Pseudomonas sp. SD705 strain (WO95 / 06720 pamphlet and WO96 / 27002 pamphlet), P. fluorescens, Pseudomonas sp. Pseudomonas lipase such as P. wiscosinensis (WO 96/12012); for example, B. subtilis (Dartois et al., 1993, Biochemica et Biophysica 113 Acta. 253-360), B.I. B. stearothermophilus (Japanese Patent Application Laid-Open No. 64/74492) or B. stearothermophilus. Bacillus lipases such as B. pumilus (WO 91/16422).

  Other examples are WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292 pamphlet, WO 95/30744 pamphlet, WO 94/25578 pamphlet, WO 95/14783 pamphlet, WO 95/22615 pamphlet, WO 97 / Lipase changes such as those described in pamphlet 04079, pamphlet WO 97/07202, pamphlet WO 00/060063, pamphlet WO 2007/0887508 and pamphlet WO 2009/109500 It is a foreign body.

  Preferred commercially available lipase enzymes include Lipolase (TM), Lipolase Ultra (TM) and Lipex (TM), Lecitase (TM), Lipolex (TM); Lipoclean (TM), Lipoprime (TM) (Novozymes A / S ). Other commercially available lipases include Lumafast (Genencor Int Inc); Lipomax (Gist-Brocades / Genencor Int Inc) and the Bacillus sp lipase from Solvay.

  Amylase: Suitable amylases (α and / or β) include those of bacterial or fungal origin. Chemically modified or protein modified mutants are included. Examples of amylases include α-amylases obtained from, for example, the genus Bacillus, such as a special strain of Bacillus licheniformis, which is described in more detail in British Patent 1,296,839. Can be mentioned.

  Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873 and WO 97/43424. In particular, variants with substitutions at one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408 and 444.

  Commercially available amylases are Steinzyme ™, Natalase ™, Duramyl ™, Termamyl ™, Fungamyl ™ and BAN ™ (Novozymes A / S), Rapidase ™, and Purastar. (Trademark) (Genencor International Inc.).

  Peroxidase / oxidase: Suitable peroxidases / oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein modified mutants are included. Examples of useful peroxidases include, for example, C.I. Peroxidases from the genus Coprinus such as C. cinereus and those described in WO 93/24618, WO 95/10602 and WO 98/15257 And its mutants.

  A commercially available peroxidase includes Guardzyme ™ (Novozymes A / S).

  Detergent enzymes may be included in the detergent composition by adding individual additives containing one or more enzymes, or by adding a complex additive containing all of these enzymes. . The detergent additives of the present invention (i.e., individual or composite additives) can be formulated, for example, as granules, liquids, slurries, and the like. Preferred detergent additive formulations are granules, especially non-dusting granules, liquids, particularly stabilized liquids or slurries.

  Non-dusting granules can be produced, for example, as disclosed in US Pat. No. 4,106,991 and US Pat. No. 4,661,452, and are optionally known in the art. It may be coated by a method. Examples of waxy coatings are poly (ethylene oxide) products (polyethylene glycol, PEG) having an average molar weight of 1000-20000; ethoxylated nonylphenol having 16-50 ethylene oxide units; 12-20 alcohols Ethoxylated fatty alcohols containing carbon atoms and having 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. An example of a film-forming coating material suitable for use with fluidized bed technology is described in British Patent No. 14835991. Liquid enzyme preparations can be stabilized, for example, by adding polyols such as propylene glycol, sugars or sugar alcohols, lactic acid or boric acid according to established methods. The protected enzyme can be prepared according to the method disclosed in EP 238,216.

Adjuncts Any detergent ingredients known in the art used in laundry detergents may also be utilized. As other optional detergent components, alone or in combination, anticorrosion agent, anti-shrink agent, anti-fouling agent, anti-wrinkle agent, disinfectant, binder, corrosion inhibitor, disintegrant / decomposition agent, dye, enzyme stabilization Agents (including boric acid, borates, CMC and / or polyols such as propylene glycol), fabric conditioners containing clay, fillers / processing aids, fluorescent whitening agents / optical brighteners, Examples include foam enhancers, foaming (soap foam) modifiers, fragrances, contaminants-suspensions, softeners, soap foam inhibitors, anti-fading agents, and wicking agents. Any formulation ingredient known in the art for use in laundry detergents can be utilized. The selection of such formulation ingredients is well within the skill of those skilled in the art.

  Dispersants—The detergent compositions of the present invention can also contain dispersants. In particular, the powder detergent may contain a dispersant. Suitable water-soluble organic materials include homo- or copolymer acids or salts thereof, wherein the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by no more than two carbon atoms. Suitable dispersants are described, for example, in Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc. It is described in.

  Anti-transfer agents—The detergent compositions of the present invention may also include one or more anti-transfer agents. Suitable polymeric dye transfer inhibitors include, but are not limited to, polyvinyl pyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinyl pyrrolidone and N-vinyl imidazole, polyvinyl oxazolidone and polyvinyl imidazole or mixtures thereof. It is done. When present in the subject compositions, the dye transfer inhibitor is about 0.0001% to about 10%, about 0.01% to about 5%, or even about 0.1% to about 5% of the composition. It can be present at a level of 3% by weight.

  Fluorescent whitening agent—The detergent compositions of the present invention will also preferably contain additional components that may change the hue of the article to be cleaned, such as a fluorescent whitening agent or an optical brightener. When present, the brightener is preferably at a level of from about 0.01% to about 0.5%. Any fluorescent whitening agent may be used in the compositions of the present invention as long as it is suitable for use in a laundry detergent composition. The most commonly used fluorescent whitening agents are those belonging to the class of diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives and bisphenyl-distyryl derivatives. Examples of fluorescent whitening agents of the diaminostilbene-sulfonic acid derivative type are: 4,4′-bis- (2-diethanolamino-4-anilino-s-triazin-6-ylamino) stilbene-2,2 4,4'-bis- (2,4-dianilino-s-triazin-6-ylamino) stilbene-2.2'-disulfonate; 4,4'-bis- (2-anilino-4) (N-methyl-N-2-hydroxy-ethylamino) -s-triazin-6-ylamino) stilbene-2,2'-disulfonate, 4,4'-bis- (4-phenyl-2,1,3 -Triazol-2-yl) stilbene-2,2'-disulfonate; 4,4'-bis- (2-anilino-4 (1-methyl-2-hydroxy-ethylamino) -s-triazine 6-ylamino) stilbene-2,2′-disulfonate and sodium 5- (2H-naphtho [1,2-d] [1,2,3] triazol-2-yl) -2-[(E) -2 -Phenylvinyl] Sodium salt of benzenesulfonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS commercially available from Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium salt of 4,4'-bis- (2-morpholino-4-anilino-s-triazin-6-ylamino) stilbene disulfonate. Tinopal CBS is the disodium salt of 2,2'-bis- (phenyl-styryl) -disulfonate. A preferred fluorescent whitening agent is the commercially available Parawhite KX manufactured by Paramount Minerals and Chemicals, Mumbai, India. Other fluorescent agents suitable for use in the present invention include 1-3-diarylpyrazolines and 7-alkylaminocoumarins. Suitable fluorescent brightener levels are from a lower level of about 0.01, 0.05, about 0.1, or even about 0.2% by weight, 0.5 or even 0.75% by weight. Including the upper limit level.

  Contaminant-Free Polymers-The detergent compositions of the present invention also include one or more types that assist in the removal of contaminants from fabrics such as cotton and polyester fabrics, particularly the removal of hydrophobic contaminants from polyester fabrics. Contaminant free polymer may be included. The contaminant free polymer may be, for example, a nonionic or anionic terephthalate-based polymer, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides (eg Chapter 7, Powdered detergents, Surfactant science series volume 71, Marcel Dek, 71). Inc.). Another type of contaminant free polymer is an amphiphilic alkoxylated oil cleaning polymer that includes a core structure and a plurality of alkoxylate groups attached to the core structure. The core structure may comprise a polyalkyleneimine structure or a polyalkanolamine structure, as detailed in WO2009 / 087523 (incorporated herein by reference). In addition, random graft copolymers are suitable contaminant free polymers. Suitable graft copolymers are described in more detail in WO 2007/138504, WO 2006/108856 and WO 2006/113314 (incorporated herein by reference). Yes. Other contaminant free polymers include modified cellulose derivatives such as those described in EP 1867808 or WO 2003/040279, both of which are hereby incorporated by reference. In particular, it is a substituted polysaccharide structure such as a substituted cellulose structure. Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides and mixtures thereof. Suitable cellulosic polymers include anionic modified cellulose, nonionic modified cellulose, cationic modified cellulose, zwitterionic modified cellulose, and mixtures thereof. Suitable cellulosic polymers include methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxylethylcellulose, hydroxylpropylmethylcellulose, ester carboxymethylcellulose and mixtures thereof.

  Anti-fouling agent-The detergent composition of the present invention also includes carboxymethyl cellulose (CMC), polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyoxyethylene and / or polyethylene glycol (PEG), homopolymers of acrylic acid , Copolymers of acrylic acid and maleic acid, and one or more anti-staining agents such as ethoxylated polyethyleneimine. The cellulosic polymer described in the above contaminant free polymer may also function as a recontamination inhibitor.

  Other suitable auxiliary materials include, but are not limited to, antishrink agents, wrinkle inhibitors, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam control agents, hydrostatic agents. Examples include lobes, fragrances, pigments, soap foam inhibitors, solvents, structurants for liquid detergents, and / or structural elasticizers.

Detergent Product Formulations The detergent composition of the present invention is, for example, a bar, a homogeneous tablet, a tablet having two or more layers, a normal or compressed powder, a granule, a paste, a gel, or a normal compression. Alternatively, it can be in any convenient form such as a concentrated liquid.

  Detergent formulation forms: forms corresponding to layers (same or different phases), pouches, mechanical dosage units.

  The pouch can be configured as a single or multiple compartments. It can be of any form, shape and material suitable for holding the composition, for example, preventing leakage of the composition such that the composition leaks from the pouch prior to contact with water. The pouch is made of a water-soluble film having an internal volume. The internal volume can be separated into a compartment of the pouch. Preferred films are polymeric materials that are preferably polymers that are formed into films or sheets. Preferred polymers, copolymers or derivatives thereof are polyacrylates and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, maltodextrin, polymethacrylate, most preferably polyvinyl alcohol copolymers and hydroxy Selected from propylmethylcellulose (HPMC). Preferably, the level of polymer in the film is at least about 60%, for example with PVA. A preferred average molecular weight will typically be from about 20,000 to about 150,000. The film is also hydrolytically degradable, such as polyactide and polyvinyl alcohol (known under the trade name M8630 commercially available from MonoSol LLC, Indiana, USA), with a blend of water soluble polymers and glycerol, ethylene glycerol, It may consist of a blend composition comprising a plasticizer such as propylene glycol, sorbitol and mixtures thereof. The pouch can include a solid laundry cleaning composition or partial component and / or a liquid cleaning composition or partial component separated by a water soluble film. The compartment for the liquid component can be different in composition from the compartment containing solids. Reference: (US Patent Application Publication No. 2009/0011970 A1)

  The detergent formulation components can be physically separated from each other by compartments in the water-soluble pouch or in different layers of the tablet. This can prevent negative storage interactions between components. Each different dissolution profile in the compartment can also delay the dissolution of selected components in the wash solution.

Form definition / features:
Liquid or gel detergents that are not in a unit dose can be aqueous, typically up to about 70% water, up to about 65% water, up to about 55% water, up to about 45% water. It may contain water, at least 20% by weight, such as up to about 35% water and up to 95% water. Other types of liquids including but not limited to alkanols, amines, diols, ethers and polyols may be included in the aqueous liquid or gel. The aqueous liquid or gel detergent may contain 0-30% organic solvent.

  Liquid or gel detergents may be non-aqueous.

Solid laundry soap The enzyme of the present invention can be added to solid laundry soap and used for laundry, fabric and / or hand washing of fabrics. The term “solid laundry soap” includes laundry soap, solid soap, composite cosmetic soap, synthetic cosmetic soap and detergent soap. The types of bar soaps generally differ in the types of surfactants they contain, and the term bar soaps includes those containing fatty acid derived soaps and / or synthetic soaps. Solid laundry soaps are in a physical form that is solid at room temperature and are not liquids, gels or powders. The term solid is defined as a physical form that does not change significantly over time. That is, if a solid material (for example, solid laundry soap) is disposed in the container, the solid material is not changed and the container in which the solid material is disposed is not filled. The solid laundry soap is typically rod-shaped, but may have another shape such as a circle or an ellipse.

Solid laundry soaps are protease inhibitors such as one or more enzymes, peptide aldehydes (or hydrosulfite adducts or hemiacetal adducts), boric acid, borates, borax and / or phenylboronic acid derivatives, For example, 4-formylphenylboric acid, one or more soaps or synthetic surfactants, polyols such as glycerine, pH control compounds such as fatty acids, citric acid, acetic acid and / or formic acid, and / or monovalent cations and A salt of an organic anion can be included, where the monovalent cation can be, for example, Na ⁺ , K ⁺ or NH ₄ ⁺ and the organic anion can be, for example, formate, acetate, citrate or It can be a lactate, so the salt of a monovalent cation and an organic anion can be Good me.

  Solid laundry soaps are also complexing agents such as EDTA and HEDP, perfumes and / or other types of fillers, surfactants such as anionic synthetic surfactants, builders, polymeric soil release agents, detergent chelates Agent, stabilizer, filler, dye, colorant, dye transfer inhibitor, alkoxylated polycarbonate, foam suppressant, organizing agent, binder, leaching agent, bleach activator, mud stain remover, anti-redeposition agent , Polymeric dispersants, whitening agents, fabric softeners, perfumes and / or other compounds known in the art.

  Solid laundry soaps include, but are not limited to, conventional solid laundry soap production equipment such as mixers, prodders (eg, two-stage vacuum prodder), extruders, cutters, logo stampers, cooling tunnels and wrapping equipment May be processed. The present invention is not limited to producing solid laundry soap in any single way. The premix of the present invention may be added to the soap at various stages of the process. For example, after preparing a premix containing soap, enzyme, optionally one or more other enzymes, protease inhibitors, and salts of monovalent cations and organic anions, the mixture is ploded. The enzyme and optionally another enzyme, for example, in liquid form, may be added simultaneously with the protease inhibitor. In addition to the mixing and probing steps, the method may further include grinding, extruding, cutting, stamping, cooling and / or lapping steps.

Granular detergent formulations Granular detergents are described in WO 09/092699, EP 1705241, EP 1382668, WO 07/001262, US Pat. No. 6,472,364, It can be formulated as described in WO 04/0774419 or WO 09/102854. Other useful detergent formulations are WO 09/124162, WO 09/124163, WO 09/117340, WO 09/117341, WO 09/124. 117342 pamphlet, WO 09/072069 pamphlet, WO 09/063355 pamphlet, WO 09/132870 pamphlet, WO 09/121757 pamphlet, WO 09/112296 pamphlet, International Publication No. 09/112298, International Publication No. 09/103822, International Publication No. 09/087033, International Publication No. 09/050026, International Publication No. 09/04712 Pamphlet, WO09 / 047126 pamphlet, WO09 / 047127 pamphlet, WO09 / 047128 pamphlet, WO09 / 021784 pamphlet, WO09 / 010375 pamphlet, International publication 09/000605 pamphlet, WO 09/122125 pamphlet, WO 09/095645 pamphlet, WO 09/040544 pamphlet, WO 09/040545 pamphlet, WO 09/024780 Pamphlet, WO09 / 004295 pamphlet, WO09 / 004294 pamphlet, WO09 / 121725 pamphlet, WO09 / 115391 pamphlet Fret, WO09 / 115392 pamphlet, WO09 / 074398 pamphlet, WO09 / 074403 pamphlet, WO09 / 068501 pamphlet, WO09 / 067770 pamphlet, International publication no. 09/021813 pamphlet, WO09 / 030632 pamphlet, WO09 / 015951 pamphlet, WO20111025615 pamphlet, WO2011016958 pamphlet, WO2011005803 pamphlet, International publication no. 2011005623 pamphlet, International Publication No. 2011005730 pamphlet, International Publication No. 2011005844 pamphlet, International Publication No. 20110 05904 pamphlet, international publication 2011005630 pamphlet, international publication 2011005830 pamphlet, international publication 2011005912 pamphlet, international publication 2011005905 pamphlet, international publication 2011005910 pamphlet, international publication 2011005813 pamphlet, international publication 20110235238. Pamphlet, international publication 20101020863 pamphlet, international publication 201020108002 pamphlet, international publication 20101011365 pamphlet, international publication 2010108000 pamphlet, international publication 2010107635 pamphlet, international publication 20100090915 pamphlet, international publication 20110033976 Pamphlet International Publication No. 20110033746 pamphlet, International Publication No. 20110033747 pamphlet, International Publication No. 20110033897 pamphlet, International Publication No. 20110033979 pamphlet, International Publication No. 2010030540 pamphlet, International publication No. 20100305541 pamphlet, International publication No. 201010030539 pamphlet, International Publication No. 20120024467 Pamphlet, International Publication No. 20120024469 Pamphlet, International Publication No. 20120024470 Pamphlet, International Publication No. 20120025161 Pamphlet, International Publication No. 20120014395 Pamphlet, International Publication No. 20110044905 Pamphlet, International Publication No. 201014585887 Pamphlet, International Publication No. Open 2010 42503 pamphlet, international publication 20101022051 pamphlet, international publication 201020102861 pamphlet, international publication 201010099997 pamphlet, international publication 20110084039 pamphlet, international publication 20110076292 pamphlet, international publication 20110069742 pamphlet, international publication 20110069718. Pamphlet, International Publication No. 20110069957, International Publication No. 20110057784, International Publication No. 20110054986, International Publication No. 2010018043, International Publication No. 2010003783, International Publication No. 2010003792, International Publication No. 20110337216 Pamphlet International publication 201020102539 pamphlet, international publication 201010118959 pamphlet, international publication 201010115813 pamphlet, international publication 2010105942 pamphlet, international publication 20101059661 pamphlet, international publication 20101059662 pamphlet, international publication 20110094356 pamphlet. International Publication No. 201100843, International Publication No. 20100078979, International Publication No. 20110072456, International Publication No. 20110069905, International Publication No. 20110076165, International Publication No. 20110072603, International Publication No. 20100066486, International Publication No. 2010 66331 pamphlet, International Publication No. 20110066632 pamphlet, International Publication No. 20110063689 pamphlet, International Publication No. 20110060821 pamphlet, International Publication No. 20110049187 pamphlet, International Publication No. 20130031607 pamphlet, International Publication No. 201010000636 pamphlet. .

Uses The present invention also relates to methods of using the proteases or compositions thereof of the present invention in dough and fabric laundry, such as household laundry washing and industrial laundry washing, and for animal feed.

  The present invention further relates to a method of using the composition for hard surface cleaning such as automatic dishwashing (ADW), car washing and industrial surface cleaning. Accordingly, in one embodiment, the present invention relates to the use of a detergent composition comprising a protease of the present invention or a protease of the present invention having sequence identity to a mature polypeptide in laundry or dishwashing. Thus, in one embodiment, the invention has protease activity and is at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, relative to the mature polypeptide of SEQ ID NO: 2, At least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77 %, At least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, At least 90% less An isolated poly having a sequence identity of 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% It relates to the use of peptides in detergents, cleaning methods and / or washing methods.

  Use in detergents. The polypeptides of the present invention can be added and thereby become a component of a detergent composition.

  The detergent composition of the present invention can be formulated, for example, as a hand-washed or mechanical laundry detergent composition containing a laundry additive composition suitable for pretreatment of soiled fabrics, and as a rinse-added fabric softener composition. Alternatively, it can be formulated as a detergent composition for use in general household hard surface cleaning operations, or it can be formulated for hand washing or machine dishwashing operations.

  In certain embodiments, the present invention provides a detergent additive comprising a polypeptide of the present invention as described herein.

Use of the Protease of the Invention in Detergents Contaminants and soils important to detergent formulations are composed of many different substances, a series of different enzymes, all of which have different substrate specificities, are laundry and It has been developed for use in detergents associated with both hard surface cleaning such as dishwashing. In order to specifically improve the soil removal in the applied cleaning process compared to the same process without enzyme, these enzymes are believed to provide an enzyme detergency benefit. Soil removal enzymes known in the art include enzymes such as carbohydrase, amylase, protease, lipase, cellulase, hemicellulase, xylanase, cutinase and pectinase.

  In one aspect, the invention provides at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67 relative to the mature polypeptide of SEQ ID NO: 2. %, At least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, At least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92 %,at least Detergent compositions and laundry of isolated polypeptides having a sequence identity of 3%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% And use in cleaning methods such as hard surface cleaning. Thus, in one aspect, the present invention demonstrates the detergency effect of various exemplary proteases of the present invention on various soils under various conditions. In a particular embodiment of the invention, the use in a detergent composition and cleaning process relates to the use of a protease according to the invention with at least one of the above soil removal enzymes.

  In a preferred embodiment of the invention, the proteases of the invention that are useful according to the invention may be combined with at least two enzymes. These additional enzymes are detailed in the “Other Enzymes” section, with at least 3, 4 or 5 enzymes being more preferred. Preferably, the enzymes have different substrate specificities such as, for example, carbolytic activity, proteolytic activity, amylolytic activity, lipolytic activity, hemicellulose degrading activity or pectin degrading activity. The enzyme combinations include, for example, the protease and amylase of the present invention, the protease and cellulase of the present invention, the protease and hemicellulase of the present invention, the protease and lipase of the present invention, the protease and cutinase of the present invention, the protease and pectinase of the present invention, Or, for example, the protease of the present invention and other soil removal enzymes, such as the protease and anti-redeposition enzyme of the present invention. More preferably, the protease of the present invention is combined with at least two other soil-removing enzymes, eg, the protease, lipase and amylase of the present invention; or the protease, amylase and pectinase of the present invention; Protease, amylase and cutinase; or protease, amylase and cellulase of the invention; or protease, amylase and hemicellulase of the invention; or protease, lipase and pectinase of the invention; or protease, lipase and cutinase of the invention Or the protease, lipase and cellulase of the present invention; or the protease, lipase and hemicellulase of the present invention. Even more preferably, the protease of the present invention may be combined with at least three other soil-removing enzymes, such as the protease, amylase, lipase and pectinase of the present invention; or the protease, amylase, lipase and cutinase of the present invention. Or a protease, amylase, lipase and cellulase of the invention; or a protease, amylase, lipase and hemicellulase of the invention. The proteases of the invention may be combined with any of the enzymes selected from a non-complete list including: carbohydrases such as amylase, hemicellulase, pectinase, cellulase, xanthanase or pullulanase, peptidases, other proteases or lipases.

  In other embodiments of the invention, the protease of the invention may be combined with one or more metalloproteases, such as M4 metalloproteases including Neutrase ™ or thermolysin. Such combinations may further include other detergent enzyme combinations outlined above.

  The cleaning process or dough handling process can be, for example, a laundry process, a dishwashing process, or a cleaning of hard surfaces such as bathroom tiles, floors, table surfaces, drains, sinks and basins. The laundry process can be, for example, household laundry, but can also be industrial laundry. Moreover, the present invention relates to a fabric and / or garment laundering process, wherein the process comprises treating the fabric with a detergent composition and a cleaning solution containing at least one protease of the present invention. The cleaning process or the dough handling process can be performed, for example, in a mechanical cleaning process or a manual cleaning process. The cleaning solution can be, for example, an aqueous cleaning solution containing a detergent composition.

  The fabric and / or clothes subjected to the cleaning, cleaning or fabric handling process of the present invention may be a conventional washable laundry, for example a home laundry. Preferably, the main products of the laundry are garments and fabrics including knitted, woven, denim, non-woven, felt, yarn and towels. The fabric is a natural cellulosic material including cotton, flax, flax, jute, ramie, sisal or coir, or artificial cellulose containing viscose / rayon, ramie, cellulose acetate fiber (tricellular), lyocell or a blend thereof It may be a cellulosic material such as a system material (eg, wood pulp derived). The fabric can also be non-cellulosic materials such as natural polyamides including wool, camel, cashmere, mohair, rabbit and silk, or synthetic polymers such as nylon, aramid, polyester, acrylic, polypropylene and spandex / elastan, or these It can be a blend and a blend of cellulosic and non-cellulosic fibers. Examples of blends are cotton and / or rayon / viscose and wool, synthetic fibers (eg polyamide fibers, acrylic fibers, polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers) and A blend with one or more associated materials such as cellulose-containing fibers (eg, rayon / viscose, ramie, flax, flax, jute, cellulose acetate fibers, lyocell).

  In recent years, there has been an increasing interest in replacing components in detergents derived from petrochemicals with renewable biological components such as enzymes and polypeptides without sacrificing cleaning performance. When the components of the detergent composition are changed, new enzyme activities or new enzymes with modified and / or improved properties compared to commonly used detergent enzymes such as proteases, lipases and amylases Is required to achieve similar or improved cleaning performance as compared to conventional detergent compositions.

  The present invention further relates to the use of the protease of the invention, which is a proteinaceous soil removal process. Proteinaceous soils can be soils such as food soils such as baby food, sebum, cacao, egg, blood, milk, ink, grass or combinations thereof.

  Typical detergent compositions contain various components in addition to enzymes, these components have different effects, and some components, like surfactants, reduce the surface tension in the detergent. Can lift, disperse, and then wash away the soil to be cleaned, other components such as bleach systems are often removed and faded by oxidation, and many bleaches are strongly sterilized It also has properties and is used for disinfection and sterilization. Still other ingredients such as builders and chelating agents soften the wash water, for example, by removing metal ions from the liquid.

  In certain embodiments, the invention relates to the use of a composition comprising a protease of the invention, wherein the enzyme composition comprises at least one or more of the following surfactants, builders, chelating agents or chelating agents. Further includes a bleach system or bleach component in the agent, laundry or dishwashing.

  In a preferred embodiment of the present invention, the amount of surfactant, builder, chelating agent or chelating agent, bleach system and / or bleach component is the surfactant, builder used without the addition of the protease of the present invention. Reduced relative to the amount of chelating agent or chelating agent, bleach system and / or bleach component. Preferably, at least one component which is a surfactant, builder, chelating agent or chelating agent, bleach system and / or bleach component is added by the protease of the present invention, such as conventional amounts of such components. 1% less than the amount of components in the system that is not used, 2% less, 3% less, 4% less, 5% less, 6% less, 7% less, 8% less, etc. % Less, 10% less, 15% less, 20% less, 25% less, 30% less, 35% less, 40% less, 45% less, 50% less, etc. Exists. In one embodiment, the protease of the present invention is used in a detergent composition, wherein the composition comprises a surfactant, builder, chelator or chelator, bleach system or bleach component, and / or It does not contain at least one component that is a polymer.

Cleaning Method The detergent composition of the present invention is ideally suited for use in laundry applications. Accordingly, the present invention includes a method of washing fabric. The method includes contacting a selected laundry with a cleaning laundry solution comprising a detergent composition according to the present invention. The fabric may include any fabric that can be laundered at the conditions used by normal consumers. This solution preferably has a pH of about 5.5 to about 9. The composition may be utilized in the solution at a concentration of about 100 ppm, preferably 500 ppm to about 15,000 ppm. The water temperature is typically about 10 ° C, about 15 ° C, about 20 ° C, about 25 ° C, about 30 ° C, about 35 ° C, about 40 ° C, about 45 ° C, about 50 ° C, about 55 ° C, about 60 ° C. C., about 65.degree. C., about 70.degree. C., about 75.degree. C., about 80.degree. C., about 85.degree. C. and about 90.degree. The water to fabric ratio is typically about 1: 1 to about 30: 1.

  In certain embodiments, the cleaning method is at a pH of about 5.0 to about 11.5, or in alternative embodiments about 5 to about 11, about 5 to about 10, about 5 to about 9, about 5 to about 8, about 5 to about 7, about 5.5 to about 11, about 5.5 to about 10, about 5.5 to about 9, about 5.5 to about 8, about 5.5 to about 7 About 6 to about 11, about 6 to about 10, about 6 to about 9, about 6 to about 8, about 6 to about 7, about 6.5 to about 11, about 6.5 to about 10, about 6. 5 to about 9, about 6.5 to about 8, about 6.5 to about 7, about 7 to about 11, about 7 to about 10, about 7 to about 9, or about 7 to about 8, and more about 6 to It is carried out at about 10.5, preferably about 5.5 to about 9, and more preferably about 6 to about 8.

  In certain embodiments, the cleaning method comprises about 1 ° dH, about 2 ° dH, about 3 ° dH, about 4 ° dH, about 5 ° dH, about 6 ° dH, about 7 ° dH, about 8 ° dH, About 9 ° dH, about 10 ° dH, about 11 ° dH, about 12 ° dH, about 13 ° dH, about 14 ° dH, about 15 ° dH, about 16 ° dH, about 17 ° dH, about 18 ° dH, About 19 ° dH, about 20 ° dH, about 21 ° dH, about 22 ° dH, about 23 ° dH, about 24 ° dH, about 25 ° dH, about 26 ° dH, about 27 ° dH, about 28 ° dH, It is performed at a hardness of about 0 ° dH to about 30 ° dH, such as about 29 ° dH, about 30 ° dH. Under typical European wash conditions, the hardness is about 15 ° dH, under typical US wash conditions, about 6 ° dH, and under typical Asian wash conditions, about 3 ° dH. .

  The present invention relates to a method for cleaning fabrics, tableware or hard surfaces using a detergent composition comprising the protease of the present invention.

  A preferred embodiment relates to a cleaning method, said method comprising: contacting an object with a cleaning composition comprising a protease of the invention under conditions suitable for cleaning said object. In a preferred embodiment, the cleaning composition is a detergent composition and the process is a laundry or dishwashing process.

  Yet another embodiment relates to a method of removing soil from a fabric, which comprises contacting the fabric with a composition comprising a protease of the present invention under conditions suitable for cleaning the object.

  In a preferred embodiment, the composition used in the above method comprises the above “other”, such as an enzyme selected from the group consisting of carbohydrase, peptidase, protease, lipase, cellulase, xylanase or cutinase, or combinations thereof. It further comprises at least one additional enzyme as described in the section “Enzymes”. In still other preferred embodiments, the composition reduces at least one or more of the following: surfactant, builder, chelating agent or chelating agent, bleach system or bleach component, or polymer component Included in a given amount.

  Also contemplated are compositions and methods for treating fabrics (e.g., for exfoliating from a dough) using one or more proteases of the present invention. Proteases can be used in any fabric processing method well known in the art (see, eg, US Pat. No. 6,077,316). For example, in one aspect, the touch and appearance of the fabric is improved by a method that includes contacting the fabric with a protease in solution. In one embodiment, the fabric is treated with the solution under pressure.

  In one embodiment, the protease is applied during or after weaving the dough, or during the desizing stage, or during one or more additional fabric processing steps. During the knitting of the fabric, the yarn is exposed to considerable mechanical stress. Prior to knitting on a mechanical loom, warp yarns are often coated with sizing starch or starch derivatives to increase their tensile strength and prevent breakage. Proteases can be applied to remove these sizing proteins or protein derivatives. After the fabric is woven, the fabric can be advanced to the desizing stage. This can be followed by one or more additional fabric processing steps. Desizing is an operation to remove sizing from the dough. After knitting, the sizing coating should be removed before further processing of the fabric to ensure uniform and wash-resistant results. There is also provided a desizing process involving enzymatic hydrolysis of sizing by the action of the enzyme.

Use at Low Temperature One embodiment of the present invention relates to a method for performing a laundry, dishwashing or industrial cleaning comprising contacting the protease to be cleaned with a surface to be cleaned, wherein said laundry, dishwashing, industrial Useful or systematic cleaning is performed at a temperature of about 40 ° C. or less. One embodiment of the invention relates to the use of a protease of the invention in a laundry, dishwashing or cleaning process, wherein the temperature in the laundry, dishwashing, industrial cleaning is about 40 ° C. or less.

  In another embodiment, the invention relates to the use of a protease of the invention in a protein removal process, wherein the temperature in the protein removal process is about 40 ° C. or less.

  The invention also relates to the use of a protease according to the invention having at least one improved property compared to Savinase (SEQ ID NO: 6) in a laundry, dishwashing or industrial cleaning process, wherein the laundry, dishwashing or The temperature in the cleaning process is performed at a temperature of about 40 ° C. or less.

  In each of the above recognized methods and uses, the cleaning temperature is about 40 ° C. or lower, about 39 ° C. or lower, such as about 38 ° C. or lower, such as about 37 ° C. or lower, such as about 36 ° C. or lower, such as about 35 ° C. or lower. About 34 ° C or less, about 33 ° C or less, about 32 ° C or less, about 31 ° C or less, about 30 ° C or less, about 29 ° C or less, about 28 ° C or less, about 27 ° C or less, etc. 26 ° C. or lower, about 25 ° C. or lower, about 24 ° C. or lower, about 23 ° C. or lower, about 22 ° C. or lower, about 21 ° C. or lower, about 20 ° C. or lower, about 19 ° C. or lower, about 18 ° C. About 17 ° C or less, about 16 ° C or less, about 15 ° C or less, about 14 ° C or less, about 13 ° C or less, about 12 ° C or less, about 11 ° C or less, about 10 ° C or less, etc. About 9 ° C or lower, about 8 ° C or lower, about 7 ° C or lower, about 6 ° C Such as lower, such as about 5 ° C. or less, such as about 4 ° C. or less, such as about 3 ° C. or less, such as about 2 ℃ or less, such as about 1 ℃ or less.

  In other preferred embodiments, the washing temperature is about 5-30 ° C, about 5-20 ° C, about 5-10 ° C, about 10-40 ° C, about 10-30 ° C, about 10-20 ° C, about 15- About 5-40, such as 40 ° C, about 15-30 ° C, about 15-20 ° C, about 20-40 ° C, about 20-30 ° C, about 25-40 ° C, about 25-30 ° C, or about 30-40 ° C It is in the range of ° C. In certain preferred embodiments, the washing temperature is about 30 ° C.

  In certain embodiments, the cryogenic cleaning method is at a pH of about 5.0 to about 11.5, or in alternative embodiments, further about 5 to about 11, about 5 to about 10, about 5 to about 9, about 5 to about 8, about 5 to about 7, about 5.5 to about 11, about 5.5 to about 10, including about 5.5 to about 9, about 5.5 to about 8 About 5.5 to about 7, about 6 to about 11, about 6 to about 10, about 6 to about 9, about 6 to about 8, about 6 to about 7, about 6.5 to about 11, about 6. 5 to about 10, about 6.5 to about 9, about 6.5 to about 8, about 6.5 to about 7, about 7 to about 11, about 7 to about 10, about 7 to about 9, or about 7 to It is carried out at about 6 to about 10.5, such as about 8, preferably about 5.5 to about 9, and more preferably about 6 to about 9.

  In certain embodiments, the low temperature cleaning method includes about 1 ° dH, about 2 ° dH, about 3 ° dH, about 4 ° dH, about 5 ° dH, about 6 ° dH, about 7 ° dH, about 8 ° dH. About 9 ° dH, about 10 ° dH, about 11 ° dH, about 12 ° dH, about 13 ° dH, about 14 ° dH, about 15 ° dH, about 16 ° dH, about 17 ° dH, about 18 ° dH , About 19 ° dH, about 20 ° dH, about 21 ° dH, about 22 ° dH, about 23 ° dH, about 24 ° dH, about 25 ° dH, about 26 ° dH, about 27 ° dH, about 28 ° dH At a hardness of about 0 ° dH to about 30 ° dH, such as about 29 ° dH, about 30 ° dH. Under typical European wash conditions, the hardness is about 15 ° dH, under typical US wash conditions, about 6 ° dH, and under typical Asian wash conditions, about 3 ° dH.

Animal feed The present invention also relates to a method of using the protease of the present invention having protease activity in animal feed, and a feed composition and feed additive containing the protease of the present invention.

  Thus, in one embodiment, the invention has protease activity and is at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, relative to the mature polypeptide of SEQ ID NO: 2, At least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77 %, At least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, At least 90% less Also has 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity The present invention relates to a feed composition containing a polypeptide and a feed additive.

  Another aspect of the present invention relates to a method for processing a protein comprising adding at least one protease of the present invention to at least one protein or a protein source such as soybean. Thus, one embodiment has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least with respect to the mature polypeptide of SEQ ID NO: 2 having protease activity. 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78% At least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, small At least one isolated poly having at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity It relates to a method for processing a protein comprising the step of adding a peptide to at least one protein or a protein source such as soybean.

  The term animal encompasses any animal, including humans. Examples of animals include non-ruminants and ruminants. Ruminants include, for example, animals such as sheep, goats, and cattle, such as beef cattle, dairy cows, and calves. In certain embodiments, the animal is a non-ruminant animal. Non-ruminant animals include monogastric animals such as pigs or pigs (including but not limited to piglets, growing pigs, and sows); poultry such as turkeys, ducks and chickens (limited) Such as but not limited to broiler chickens, laying hens; horses (including but not limited to hot blood, cold blood and warm blood), calves; and fish (including but not limited to salmon, trout, Tilapia, catfish and carp; and crustaceans (including, but not limited to, shrimp and prawns).

  The term feed or feed composition means any compound, preparation, mixture or composition that is suitable for or intended for consumption by animals.

  For use according to the invention, the animal can be given the protease before feeding, after feeding or simultaneously. The latter is preferred.

  In certain embodiments, the protease is clearly defined in a form that is added to the feed or when contained in the feed additive. “Clearly defined” means that the protease preparation is at least 50% pure as determined by size exclusion chromatography (see Example 12 of WO 01/58275). . In another specific embodiment, the protease preparation is at least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95% pure as determined by this method.

  A clearly defined protease preparation is advantageous. For example, it is much easier to accurately administer to a feed a protease that is substantially free of other interfering or contaminating proteases. The term “administering”, to be precise, refers to the ability to optimize the dosage based on the desired effect, particularly with the aim of obtaining consistent and consistent results.

  However, for use in animal feed, the protease need not be so pure; for example, it may contain another enzyme, in which case it can be referred to as a protease preparation.

  Protease preparations can be used to produce one or more intermediate compositions such as (a) added directly to the feed (or used directly in the protein processing step) or (b) a feed additive or premix. These can later be added to the feed (or used in the treatment step). The aforementioned purity refers to the purity of the original protease preparation, whether used according to either (a) or (b) above.

  Protease preparations with the aforementioned degree of purity can be obtained in particular using recombinant production methods, but are not so easily obtained, and when produced by conventional fermentation methods. Will be subject to greater batch-to-batch variation.

  Of course, such protease preparations may be mixed with other enzymes.

  The protein may be animal protein such as animal meat and bone meal, feather meal, and / or fish meal; or may be vegetable protein.

  As used herein, the term “plant protein” refers to any compound, composition, preparation or mixture obtained from a plant or containing at least one protein (including modified proteins and protein derivatives) derived from a plant. Point to. In certain embodiments, the protein content of the plant protein is at least 10, 20, 30, 40, 50, or 60% (w / w).

  Plant proteins include plant protein sources such as legumes and cereals, for example, Fabaceae (Leguminosae), Brassicaceae, Chenopodiaceae, and Poaceae For example, it may be obtained from soybean meal, houched bean meal, rapeseed meal and the like.

  In certain embodiments, the vegetable protein source is material from one or more plants such as Fabaceae, for example, soybeans, waxy beans, peas or kidney beans.

  In another specific embodiment, the plant protein source is material from one or more plants, such as Chenopodiaceae, for example, beet, sugar beet, spinach or quinoa.

  Other examples of vegetable protein sources include rapeseed, sunflower seeds, cotton seeds, and cabbage.

  Soybean is a preferred vegetable protein source.

  Other examples of plant protein sources include grains such as barley, wheat, rye, oats, corn (corn), rice, triticale, and sorghum.

  In a particular embodiment of the processing method, the protease of interest is one that affects (or acts on, or solubilizes) a protein, such as a plant protein or protein source. To accomplish this, the protein or protein source is typically suspended in a solvent, for example an aqueous solvent such as water, and the pH and temperature values are adjusted while paying close attention to the properties of the enzyme of interest. Adjust. For example, the treatment is performed at a pH value where the actual protease activity is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% You can do it. Similarly, for example, at a temperature at which the actual protease activity is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% May be implemented. The above activity ratios are relative to maximum activity. The enzyme reaction may continue until the desired result is achieved, and then may or may not be stopped, for example, by inactivating the enzyme by a heat treatment step.

  In another specific embodiment of the treatment method of the present invention, the protease action is sustained, for example by adding the protease to the protein, but later at a desired time when suitable hydrolysis conditions are established. Or so-called switch on its hydrolysis action until any enzyme inhibitor is inactivated or whatever other means to postpone the action of the enzyme are considered to have been used. It means not.

  In one aspect, the treatment is a pretreatment of animal feed or protein used for animal feed. That is, the protein is hydrolyzed before ingestion.

  The term “improving the nutritional value of animal feed” means improving protein availability, resulting in increased protein extraction, higher protein yield, and / or improved protein utilization. Thus, increasing feed nutritional value also increases protein and amino acid digestibility and / or improves growth rate and / or weight gain and / or animal feed conversion (weight of feed consumed relative to weight gain) .

  The protease is either in the form of a relatively pure protease or a mixture with other ingredients intended for addition to animal feed (ie in the form of animal feed additives such as so-called premixes for animal feed). Can be added to the feed.

  In another aspect of the invention, the invention relates to compositions for use in animal feed, such as animal feed and animal feed additives, eg, premixes.

  Besides the protease of the present invention, the animal feed additive of the present invention comprises at least one fat-soluble vitamin, and / or at least one water-soluble vitamin, and / or at least one trace metal, and / or at least Contains one type of element.

  In addition, as optional feed additives, colorants such as carotenoids such as β-carotene, astaxanthin and lutein; stabilizers; growth-improving additives and aroma compounds / flavors such as creosole, anethole, deca, undeca And / or dodeca-lactone, ionone, iron, gingerol, piperidine, propylidene fatalide, butylidene phthalide, capsaicin and / or tannin; antimicrobial peptide; polyunsaturated fatty acid (PUFA); active oxygen producing species; A support may also be used, for example, 40-50 wt% wood fiber, 8-10 wt% stearin, 4-5 wt% curcuma powder, 4-58 wt% rosemary powder. 22-28% limestone, 1-3% rubber (gum arabic) Etc.), it may contain 5-50% by weight of sugar and / or starch and 5-15% by weight of water.

  The feed or feed additive of the present invention also includes phytase (EC 3.1.3.8 or 3.1.3.26); xylase (EC 3.2.1.8); galactanase (EC 3.2.1. 89); α-galactosidase (EC 3.1.2.22); additional protease, phospholipase A1 (EC 3.1.32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1.1.5). ); Phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4); amylase, for example α-amylase (EC 3.2.1.1); and / or β-glucanase (EC 3. 2.1.4 or EC 3.2.1.6) may be included.

  In certain embodiments, these other enzymes are clearly defined (as described above for protease preparations).

  Examples of antibacterial peptides (AMP) include CAP18, leucosin A, tritolepticin, protegrin-1, tanatine, defensin, lactoferrin, lactoferricin, nobispyrine (Robert Lehrer, 2000) and other bispyrins, plectins, and statins. Yes, including the compounds and polypeptides described in WO 03/0444049 and WO 03/048148, and variants or fragments of the above substances that retain antibacterial activity.

  As disclosed in WO 94/01459 and WO 02/090384, examples of antifungal polypeptides (AFP) include Aspergillus giganteus peptide, and Aspergillus niger There are (Aspergillus niger) peptides, and variants and fragments thereof that retain antifungal activity.

  Examples of polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids such as arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid and gamma linoleic acid.

  Examples of reactive oxygen producing species include perborate, persulfate, or percarbonate; and enzymes such as oxidase, oxygenase or synthase.

  Fat-soluble and water-soluble vitamins and trace metals usually form part of a so-called premix intended for addition to feed, while macroelements are usually added individually to the feed. Any of these composition types are feed additives of the present invention if enriched with the protease of the present invention.

  In certain embodiments, the animal feed additive of the present invention is 0.01 to 10.0% in animal feed; more specifically 0.05 to 5.0%; or 0.2 to 1.0. % (% Means the number of grams of additive per 100 g of feed) shall be included (or indicated as containing). This is especially true for premixes.

  The following is a non-limiting list of these example ingredients.

  Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E, and vitamin K, such as vitamin K3.

  Examples of water-soluble vitamins are vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and pantothenate, such as Ca-D-pantothenate.

  Examples of trace metals are manganese, zinc, iron, copper, iodine, selenium, and cobalt.

  Examples of macroelements are calcium, phosphorus and sodium.

  The required nutrient levels of these ingredients (illustrated for poultry and piglets / pigs) are listed in Table A of WO 01/58275. The nutrient requirement means that these ingredients should be supplied in the feed at the indicated concentrations.

  In another aspect, the animal feed additive of the present invention comprises at least one of the individual components specified in Table A of WO 01/58275. “At least one” means any one or more, one, two, or three, or four to all thirteen or all fourteen individual components. More specifically, the at least one individual component is present in an amount such that it provides a feed concentration within the range indicated in the fourth, fifth, or sixth column of Table A. Contained in the additive.

  In yet another embodiment, the animal feed additive of the present invention preferably provides the following feed concentrations within the ranges specified in Table 1 below (for piglet feed and broiler feed respectively): Contains at least one of the vitamins.

  The invention also relates to animal feed compositions. Animal feed compositions or feeds have a relatively high protein content. Poultry and pig feed can be characterized as indicated in Table B, columns 2-3 of WO 01/58275. Fish feed can be characterized as indicated in the fourth column of this Table B. Furthermore, such fish feed has a crude fat content of 200-310 g / kg.

  WO 01/58275 is consistent with US 09/793344, which is incorporated herein by reference.

  The animal feed composition of the present invention has a crude protein content of 50-800 g / kg and further comprises at least one protease as claimed herein.

  Additionally or alternatively, in another embodiment (instead of the crude protein content indicated above), the animal feed composition of the invention has a metabolic energy content of 10-30 MJ / kg; and / or 0.1-200 g / kg Calcium content; and / or available phosphorus content of 0.1-200 g / kg; and / or methionine content of 0.1-100 g / kg; and / or 0.1-150 g / kg of methionine and cysteine Content; and / or a lysine content of 0.5 to 50 g / kg.

  In certain embodiments, the content of metabolic energy, crude protein, calcium, phosphorus, methionine, methionine and cysteine, and / or lysine is in ranges 2, 3, 4 or 5 of Table B of WO 01/58275. (R. 2 to 5) is within one of the ranges.

  The crude protein is calculated as nitrogen (N) multiplied by a factor of 6.25, ie crude protein (g / kg) = N (g / kg) × 6.25. Nitrogen content is determined by the Kjeldahl method (AOAC, 1984, Official Methods of Analysis 14th ed., Association of Official Chemicals, Washington DC).

  Metabolic energy can be calculated based on the following: NRC publication Nutrient requirements in spine, 9th edition 1988, Subcommittee on Pig Nutrition, Committee on Animal Nutrition, Agricultural Conference, National Research Council. National Academy Press, Washington, D.C. C. , Pp. 2-6, as well as the European Table of Energy Values for Portal Feed-stuffs, Spelderholt center for power research and extension, 7361 DA Beebergen. Grafisch bedrijf Ponsen & loijen bv, Wageningen. ISBN 90-71463-12-5.

  The feed content of calcium, available phosphorus and amino acids in complete animal feeds can be found in Veevedertabel 1997, gevevens over chemische samenstelling, verterbaarheid en vöwerwärderwärverwedwerw, vendrwedelwerden, centrwerd. Calculate based on feed tables such as ISBN 90-72839-13-7.

  In certain embodiments, the animal feed composition of the present invention comprises at least one vegetable protein as defined above.

  The animal feed composition of the present invention may also include animal proteins such as animal meat and bone meal, feather meal, and / or fish meal, typically in an amount of 0-25%. The animal feed composition of the present invention may further comprise a dry distilled cereal residue (DDGS) with soluble material, typically in an amount of 0-30%.

  In yet another specific embodiment, the animal feed composition of the invention comprises 0-80% corn; and / or 0-80% sorghum; and / or 0-70% wheat; and / or 0 70% barley; and / or 0-30% oats; and / or 0-40% soybean meal; and / or 0-25% fish meat; and / or 0-25% animal meat and bone meal; and / or Contains 0-20% whey.

  Animal feed can be produced, for example, as mash feed (non-pelletized) or pelleted feed. Typically, the ground feed material is mixed and a sufficient amount of essential vitamins and minerals are added according to the specifications for the target species. The enzyme can be added as a solid or liquid enzyme preparation. For example, in the case of mash feed, the solid or liquid enzyme preparation may be added before or during the material mixing step. In the case of pelleted feed, a (liquid or solid) protease / enzyme preparation may also be added before or during the material mixing step. Typically, the liquid protease / enzyme preparation is added after the pelleting step. Moreover, you may contain an enzyme in a feed additive or a premix.

  The final enzyme concentration in the feed is in the range of 0.01 to 200 mg enzyme protein per kg feed, for example in the range 0.5 to 25 mg enzyme protein per kg feed.

  The protease should, of course, be added in an effective amount, i.e. an amount suitable to improve feed hydrolysis, digestibility, and / or improve nutritional value. Currently, the following amounts (dose ranges): 0.01-200; 0.01-100; 0.5-100; 1-50; 5-100; 10-100; 0.05-50; It is contemplated to administer the enzyme at one or more of 10-10, all of these ranges are mg (ppm) protease protein per kg of feed.

  To determine mg of protease protein per kg of feed, after purifying the protease from the feed composition, the specific activity of the purified protease is determined using the relevant assay (see Protease activity, substrate and assay section) . Using the same method, the protease activity of the feed composition itself is also determined, and based on these two measurements, the mg protease protein unit volume per kg of feed is calculated.

  The same principle applies to determine the protease protein mg in the feed additive. Of course, if a sample of the protease used to prepare the feed additive or feed is available, the specific activity is determined from this sample (there is no need to purify the protease from the feed composition or additive).

  The following examples further illustrate the present invention, but they should not be construed as limiting the scope of the invention.

Materials and Methods Wash Assay Automated Laundry Automated Mechanical Stress Assay (AMSA)
To evaluate the cleaning performance in the laundry, a laundry experiment is performed using an automated mechanical stress assay (AMSA). AMSA can test the cleaning performance of large volumes of small volume enzyme-detergent solutions. The AMSA plate has a number of slots for test solutions and a lid that pushes the dough, the laundry sample to be washed, firmly into all slot openings. During the cleaning time, the plate, test solution, dough and lid are shaken vigorously to bring the test solution into contact with the dough and mechanical stress is applied with regular periodic vibration. For further explanation see WO 02/42740, in particular the paragraph “Special method emblems” on pages 23-24.

  The cleaning performance is measured as the brightness of the washed fabric color. Luminance can also be expressed as the intensity of reflected light from the sample when illuminated with white light. If the sample is dirty, the intensity of the reflected light is lower than that of a clean sample. Therefore, it is possible to measure the cleaning performance using the intensity of the reflected light.

  The color measurement is performed with a professional flat scanner (Kodak iQsmart, Kodak, Midtager 29, DK-2605 Bendby, Denmark) used to take an image of the washed fabric.

In order to extract light intensity values from the scanned image, the 24-bit pixel values from the image are converted to red, green and blue (RGB) values. The intensity value (Int) is calculated by adding the RGB values together as a vector and then taking into account the length of the resulting vector.

  The test material was Center For Testmaterials BV, P.M. O. Box 120, 3133 KT Vladingen, the Netherlands and EMPA Testerials AG, Moevenstrasse 12, CH-9015 St. Obtained from Gallen, Switzerland.

Protease Assay A kinetic Suc-AAPF-pNA assay was used to obtain pH-activity profiles and pH stability profiles.

  A Protazyme AK (crosslinked dry casein) assay was used to obtain temperature-activity profiles at pH 6.5 and pH 9.

1) Suc-AAPF-pNA assay:
pNA substrate: Suc-AAPF-pNA (Bachem L-1400).
Temperature: Room temperature (25 ° C)
Assay buffer: 100 mM succinic acid, 100mM HEPES, 100mM CHES, 100mM CABS, 1mM CaCl 2, 150mM KCl, pH value 0.01% Triton X-100 (HCl or NaOH 2.0, 3.0, 4.0 , 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0).

20 μl protease (diluted with 0.01% Triton X-100) was mixed with 100 μl assay buffer. The assay was started by adding 100 μl pNA substrate (50 mg dissolved in 1.0 ml DMSO and further diluted 45-fold with 0.01% Triton X-100). As a measure of protease activity, the increase in OD ₄₀₅ was monitored.

2) Protazyme AK assay:
Substrate: Protazyme AK tablet (cross-linked and stained casein; manufactured by Megazyme)
Temperature: controlled (assay temperature).
Assay buffer: 100 mM succinic acid, 100mM HEPES, 100mM CHES, 100mM CABS, 1mM CaCl 2, 150mM KCl, 0.01% Triton X-100, pH7.0.

Protazyme AK tablets were suspended by gentle stirring in 2.0 ml of 0.01% Triton X-100. 500 μl of this suspension and 500 μl assay buffer were aliquoted into Eppendorf tubes and placed on ice. 20 μl protease sample (diluted with 0.01% Triton X-100) was added. The assay was initiated by transferring the Eppendorf tube to an Eppendorf thermomixer set at the assay temperature. Tubes were incubated for 15 minutes with an Eppendorf thermomixer at maximum shaking rate (1400 rpm). Incubation was stopped by returning the tube to an ice bath. The tube was then centrifuged for several minutes with ice-cold centrifugation and 200 μl of the supernatant was transferred to a microtiter plate. OD ₆₅₀ was read as a measure of protease activity. The assay included a buffer blind (instead of enzyme).

3) Suc-AAPX-pNA assay:
pNA substrate: Suc-AAPA-pNA (Bachem L-1775)
Suc-AAPR-pNA (Bachem L-1720)
Suc-AAPD-pNA (Bachem L-1835)
Suc-AAPI-pNA (Bachem L-1790)
Suc-AAPM-pNA (Bachem L-1395)
Suc-AAPV-pNA (Bachem L-1770)
Suc-APL-pNA (Bachem L-1390)
Suc-APE-pNA (Bachem L-1710)
Suc-AAPK-pNA (Bachem L-1725)
Suc-AAPF-pNA (Bachem L-1400)
Temperature: Room temperature (25 ° C)
Assay buffer: 100 mM succinic acid, 100mM HEPES, 100mM CHES, 100mM CABS, 1mM CaCl 2, 150mM KCl, 0.01% Triton X-100, pH value 9.0.

20 μl protease (diluted with 0.01% Triton X-100) was mixed with 100 μl assay buffer. The assay was started by adding 100 μl pNA substrate (50 mg dissolved in 1.0 ml DMSO and further diluted 45-fold with 0.01% Triton X-100). As a measure of protease activity, the increase in OD ₄₀₅ was monitored.

Soybean-corn meal assay (SMM assay)
Protease activity on soybean-corn meal at pH 3, 4, 5, 6, and 7 To obtain protease activity profiles at pH 3-7, an endpoint assay using soybean-corn meal as a substrate was used.

Assay buffer: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CAPS, 1 mM CaCl ₂ , 150 mM KCl, 0.01% Triton X-100 (10 ml assay buffer 1 g soybean-corn meal (30:70 ratio) ) Adjusted to pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 with HCl or NaOH) .
After mixing 2 mL soybean-corn meal slurry for 30 minutes, protease is added and incubation is performed at 40 ° C. for 3 hours (500 rpm). The protease is 100 μl of 100 mM sodium acetate (NaAc) buffer (9.565 g / l NaAc, 1.75 g / l acetic acid, 5 mM CaCl ₂ , 0.01% BSA, 0.01% Tween 20, pH 6.0). Use to add. Supernatant was collected after centrifugation (10 min, 4000 rpm, 0 ° C.) and essentially Nielsen et al. (Nielsen, PM, Petersen, D, Dampmann, C. Improved method for determining the food quality of JF, F1). , 66: 642-646), and the protein activity is determined using a colorimetric assay based on the o-phthalto-dialdehyde (OPA) method. Since this assay detects free α-amino groups, protease activity can be measured as an increase in absorbance. The first 500 μl of each supernatant is filtered through a 100 kDa Microcon filter by centrifugation (60 minutes, 11,000 rpm, 5 ° C.). After diluting the sample 10-fold in deionized water, 25 μl of each sample is loaded into a 96 well microplate (repeat 5 times). Finally, 200 μl of OPA reagent is dispensed into all wells, the plate is shaken (10 seconds, 750 rpm) and the absorbance is measured at 340 nm. The level of protease activity is calculated as the difference in absorbance between the enzyme treated sample and the blank sample and is expressed as "ODx dilution factor".

In Vitro Digestion Assay In a design designed to stimulate digestion in monogastric animals, an in vitro digestion assay was used to assess the effect of proteases on the feed substrate (soybean-corn meal). The incubation step consists of a digestion step in the stomach with pH 3 porcine pepsin (SP7000, Sigma-Aldrich, St. Louis, MO, USA), followed by incubation in the short duodenum at pH 3.8 and pancreatin at pH 7.0 (8 × USB , P-7545, Sigma-Aldrich, St. Louis, MO, USA).

In vitro digestion was performed using an automated system based on a Gilson liquid handler (Biolab, Denmark). For each sample, 0.8 g of feed was weighed into a test tube and all the test tubes were placed in a liquid handler (40 ° C., 500 rpm). Solution addition and pH measurements were performed automatically. At 0 minutes, 4.1 mL HCl (24 mM CaCl ₂ ) was added to achieve pH 3.0 in solution. At 30 minutes, 0.5 mL HCl (24 mM CaCl ₂ , 3000 U pepsin / feed) and 100 μl of 100 mM sodium acetate buffer (258.6 g NaAc, 0.57% acetic acid, pH 6.0 per liter) were added. At 90 minutes, 900 μL NaOH is added to achieve a pH of about 3.8, and at 120 minutes, 400 μL of 1 M NaHCO ₃ solution containing 6.5 mg pancreatin per gram of feed is added to the solution. This resulted in a pH of 6.8. The pH was measured at 30, 60, 90, 115, 120 and 180 minutes. Test protease was added using 100 μl NaAc buffer at 30 minutes.

Essentially according to Nielsen et al. (Nielsen, PM, Petersen, D, Dampmann, C. Improved method for determining food protein of hydrissis. J Food Sci, 2001-666: 642-646A). A method-based colorimetric assay is used to determine the degree of protein hydrolysis (DH). Since this assay detects free α-amino groups, protease activity can be measured as an increase in absorbance. The first 500 μl of each supernatant is filtered through a 100 kDa Microcon filter by centrifugation (60 minutes, 11,000 rpm, 5 ° C.). After the samples are diluted 100-fold in deionized water, 25 μl of each sample is loaded into a 96-well microplate (repeated 5 times). Finally, 200 μl of OPA reagent is dispensed into all wells, the plate is shaken (10 seconds, 750 rpm) and the absorbance is measured at 340 nm. The percentage (%) of cleaved peptide bonds (DH) was calculated as follows:
DH (%) = 100 × h / h _tot
( _Where h _tot is the total number of peptide bonds per protein equivalent and h is the number of hydrolyzed bonds). The calculation of h _tot is based on the amino acid sequence of the raw material. In this test, the value for soybean was used according to Adler-Nissen (1986) (7.8 g equivalent per kg protein). H in the OPA method is expressed as:
h = (serine-NH ₂ -β) / α meqv / g protein (where α = 0.970, β = 0.342 according to Adler-Nissen (1979)). Serine -NH ₂ is calculated as follows:
Serine-NH ₂ = (OD _blank -OD _sample ) / (ODN _standard- OD _blank ) × 0.9516 meqv / L × 0.1 × 100 / X × P
(Wherein serine-NH ₂ = megv serine-NH ₂ / g protein; X = g sample; P =% protein in the sample, and 0.1 is the sample amount in liters (L)).

Statistics: Statistical analysis of the registered parameters was performed using the analysis of variance (ANOVA) method, and the mean values were compared using the Student t test (α = 0.05) provided by the ANOVA method ( SAS, JMP (registered trademark) 5 Administrative Guides to Annual Licensed Windows, Mackintosh, and Linux Versions, Release 5.1, SAS Institute, Cary, NC.

Lines Saccharothrix australiensis DSM 43800, Deutsche Sammlung von Microorganismen und Zellkulturen GmbH, available from Braunschweig, Germany. This line was originally collected from Australian soil.

Media and solutions LB plates consisted of 10 g Bacto-Tryptone, 5 g yeast extract, 10 g sodium chloride, 15 g Bacto-agar, and up to 1 liter deionized water.

  The LB medium consisted of 10 g Bacto-Tryptone, 5 g yeast extract, and 10 g sodium chloride, and up to 1 liter deionized water.

Example 1: DNA preparation and DNA sequencing of the Saccharothrix australiensis genome The chromosomal DNA of Saccharotrix australiensis, QIAamp DNA Blood MiniKinQ , Germany). 5 μg of chromosomal DNA from each line was sent to FASTERIS SA (Switzerland) for genomic sequencing. The genome was sequenced by Illumina Sequencing. Genomic sequences were analyzed for secreted S1 protease and S1 protease (SEQ ID NO: 1 / SEQ ID NO: 2) was identified.

Example 2: Expression of S1 protease from Saccharothrix australiensis Based on the nucleotide sequence identified as SEQ ID NO: 1, a synthetic gene having SEQ ID NO: 3 was generated as Gene Art (GENEART AG BioPark, Josef- Engert-Str. 11, 93053, Regensburg, Germany). The synthetic gene was subcloned into the Bacillus expression vector as described in Example 1 of PCT / European Patent No. 2011/064585 using Clal and Mulul restriction sites. Transformants were selected on LB plates supplemented with 6 μg chloramphenicol per ml. Recombinant Bacillus subtilis clones containing the integrated expression construct were selected and this was added with a 500 mL baffle containing 100 ml of casein medium supplemented with 34 mg / l chloramphenicol on a rotary shaker. Incubated in Erlenmeyer flasks. The clones were cultured at 30 ° C. for 5 days. The supernatant containing the enzyme was collected and the enzyme was purified as described in Example 3.

Example 3: Purification of S1 protease from Saccharothrix australiensis The culture broth from Example 2 was centrifuged (20000 xg, 20 min) and the supernatant carefully decanted from the precipitate. did. The supernatant was filtered through a Nalgene 0.2 μm filtration unit to remove remaining Bacillus host cells. The 0.2 μm filtrate was transferred to 50 mM H ₃ BO ₃ , 20 mM CH ₃ COOH / NaOH, 1 mM CaCl ₂ , pH 4.5 on a G25 Sephadex column (manufactured by GE Healthcare). The enzyme transferred to G25 Sephadex was added to an SP-sepharose FF column (manufactured by GE Healthcare) equilibrated with 50 mM H ₃ BO ₃ , 20 mM CH ₃ COOH / NaOH, 1 mM CaCl ₂ , pH 4.5. After washing the column carefully with equilibration buffer, the protease was eluted with a linear NaCl gradient (0 → 0.5 M) in the same buffer at 5 column volumes. Fractions from the column were analyzed for protease activity (using a Suc-AAPF-pNA assay at pH 9). The protease peak is pooled and diluted 10-fold with deionized water to reduce sample conductivity and then equilibrated with 50 mM H ₃ BO ₃ , 20 mM CH ₃ COOH / NaOH, 1 mM CaCl ₂ , pH 4.5 Added to a prepared SOURCE S column (manufactured by GE Healthcare). After washing the column thoroughly with equilibration buffer, the protease was eluted with a linear NaCl gradient (0 → 0.5 M) in the same buffer at 10 column volumes. Fractions from the column were analyzed for protease activity (using the Suc-AAPF-pNA assay at pH 9) and the active fractions were further analyzed by SDS-PAGE. Fractions in which only one band was seen on a Coomassie stained SDS-PAGE gel were pooled as purified product and used for further characterization.

Example 4: Characterization of S1 protease from Saccharothrix australiensis Using the Suc-AAPF-pNA assay, pH-activity profile and pH-stability profile (2 at the stated pH value) Residual activity after time) was obtained. For pH-stability profiles, proteases were diluted 10-fold with different assay buffers to the pH values of these buffers and then incubated at 37 ° C. for 2 hours. Following incubation, the pH of the protease incubation was shifted to the same pH value prior to assaying for residual activity by dilution with pH 9.0 assay buffer. A temperature-activity profile at pH 7.0 was obtained using the Protazyme AK assay. To obtain the P1-specificity of the enzyme at pH 9.0, a Suc-AAPX-pNA assay and 10 different Suc-AAPX-pNA substrates were used.

  The results are shown in Tables 3-6 below. These results are compared with protease 10R (SEQ ID NO: 7). With respect to Table 3, the activity is relative to the optimum pH for the enzyme. Regarding Table 4, the activity is the residual activity on the sample, which was maintained under stable conditions (5 ° C., pH 9.0). With respect to Table 5, the activity is relative to the optimum temperature at pH 7.0 or pH 6.5 for the enzyme. With respect to Table 6, the activity is against the best substrate for the enzyme (Suc-AAPF-pNA).

Other features S1 protease from Saccharothrix australiensis was inhibited by PMSF. The determination of the N-terminal sequence was: IDVIGGN (SEQ ID NO: 4). The relative molecular weight determined by SDS-PAGE was approximately M _r = 21 kDa. The molecular weight determined by intact molecular weight analysis was 18746.9 Da.
Mature sequence (from MS-EDMAN data):
The molecular weight calculated from this mature sequence was 18746.5 Da.

Example 5: AMSA cleaning with S1 protease from Saccharothrix australiensis Two different liquid stains and one powder detergent were used for two different industrial soils. At the washing temperature, the washing performance of S1 protease from Saccharothrix australiensis was tested using an automatic mechanical stress assay (Automatic Mechanical Stress Assay). The experiment was performed using the detergent compositions and fabric breaks listed in Table 2 and the experimental conditions specified in Table 7 below, as described in AMSA for the laundry method using a single cycle cleaning method.

The hardness of the water was adjusted to 15 ° dH by adding CaCl ₂ , MgCl ₂ and NaHCO ₃ (Ca ²⁺ : Mg ²⁺ : CO ₃ ⁻ = 4: 1: 7.5) to the test system. After washing, the dough was rinsed with tap water and dried.

  From these results, it can be seen that the detergent containing S1 protease derived from Saccharothrix australiensis is more effective in removing soils than the detergent containing no protease. S1 protease from Saccharothrix australiensis is effective at removing blood and egg soils at 20 ° C.

  From these results, it can be seen that the detergent containing S1 protease derived from Saccharothrix australiensis is more effective in removing soils than the detergent containing no protease. S1 protease from Saccharothrix australiensis is effective at removing blood, eggs and milk dirt at 40 ° C.

Example 6: Evaluation of the stability of S1 protease from Saccharothritus australiensis in liquid detergents using AMSA Automated Mechanical Stress Assay at two wash temperatures Was used to test the cleaning performance of detergents containing proteases to test the stability of S1 protease from Saccharothritus australiensis in detergents. Three different stability conditions were tested as follows:
Protease was added to the detergent composition just prior to washing;
Protease was preincubated with detergent for 48 hours at 25 ° C; and before the start of washing, protease was preincubated in detergent solution for 30 minutes at 40 ° C.

  The experiment was carried out as described in Table 2 and the detergent compositions and fabric scraps listed in Table 2, as described in the Automatic Mechanical Stress Assay AMSA for the laundry method using a single cycle cleaning method. It was carried out using the experimental conditions specified in the inside.

Water hardness was adjusted to 15 ° dH by adding CaCl ₂ , MgCl ₂ and NaHCO ₃ (Ca ²⁺ : Mg ²⁺ : CO ₃ ⁻ = 4: 1: 7.5) to the test system. After washing, the dough was rinsed with tap water and dried.

  From these results, the detergent containing S1 protease derived from Saccharothrix australiensis has a cleaning performance after storage for 48 hours at 25 ° C. in a liquid detergent. It turns out that it is the same as the fresh enzyme. From this, it is clear that S1 protease derived from Saccharothrix australiensis exhibits detergent stability under these conditions.

  Furthermore, these results indicate that the detergent containing S1 protease from Saccharothritus australiensis has a cleaning performance after pre-incubation of the washing solution at 40 ° C. for 30 minutes just before washing. It turns out that it is the same as the washing | cleaning liquid prepared by adding an enzyme to a detergent. From this, it is clear that S1 protease derived from Saccharothrix australiensis shows stability during washing under these conditions.

Example 7: Protease Activity Against Soybean-Corn Meal Assay The results obtained by performing the aforementioned soybean-corn meal assay are shown in Table 12 below. The proteolytic activity of S1 protease from Saccharothrix australiensis on soybean-corn meal increases as the pH increases from pH 3 to pH 7, and the activity at pH 6-7 is reduced to protease 10R ( As high as the protease 10R strain from Nocardiopsis sp., Disclosed in WO 88/03947), which is the S1 protease from Saccharothrix australiensis, It shows that it has the same effect on proteolysis in the intestines of pigs and poultry around pH 7 as this commercial product.

  FIG. 1 shows the activity of S1 protease from Saccharothrix australiensis on soybean-corn meal compared to 10R protease.

Example 8: In Vitro Digestion Assay An in vitro digestion assay was performed as described above to compare S1 protease from Saccharothrix australiensis with protease 10R. The results are shown in Table 13 below. S1 protease from Saccharothrix australiensis increased the degree of proteolysis in the sample to the same level as protease 10R, because two proteases were present in soybean-corn feed It is shown to be equally effective in hydrolyzing proteins.

Example 9: Proteolytic activity against trout, gall and ileal digest from broiler chickens Collect trout, gizzard and ileal digest from 21 day old broiler chickens fed corn-soy feed; This was freeze-dried and ground using a small coffee mill. The ground sample was suspended in the following buffer (47% w / v) and allowed to hydrolyze overnight at 4 ° C. (no stirring):
Agate buffer: 100 mM HEPES, 1 mM CaCl ₂ · 2H ₂ O, 150 mM KCl, 0.01% Triton X-100, adjusted to pH 5 with HCl Ginger buffer: 100 mM succinic acid, 1 mM CaCl ₂ · 2H ₂ O, 150 mM KCl, 0.01% Triton X-100, adjusted to pH 1.67 using HCl Ileum buffer: 100 mM HEPES, 1 mM CaCl ₂ · 2H ₂ O, 150 mM KCl, 0.01% Triton X-100, HCl Use to adjust to pH 7.2

The resulting pH was pH 5 in that sample; pH 3 in the sandbag sample; and pH 7 in the ileum sample. The suspension was heated to 40 ° C. and 1 ml was suspended in a test tube maintained at 40 ° C. Three tubes representing the blank (T ₀ ) were immediately centrifuged (3000 × g, 0 ° C., 10 minutes) to freeze the supernatant. Any enzyme (200 mg) in 50 μL of 100 mM sodium acetate buffer (9.565 g / l NaOAc, 1.75 g / l acetic acid, 5 mM CaCl ₂ , 0.01% BSA, 0.01% Tween 20, pH 6.0) (Enzyme protein / kg substrate) or blank sample, sodium acetate buffer only (50 μL) was added to the tube, then the umbilical and ileal samples were 3 hours (T ₃ ), and the sandbag samples were 1 hour (T ₁ And incubation at 40 ° C. with stirring (500 rpm). Samples were centrifuged (3000 × g, 0 ° C., 10 minutes) and the supernatant was collected and frozen. Proteolytic activity was determined by analyzing primary amines using an o-phthaldialdehyde (OPA) assay. The results are shown in Table 14. Digest type (U thereof, gizzard and ileum) for each, and the blank T ₀ samples, there was a significant difference in the level of soluble primary amine in the blank samples incubated 1 or 3 hours. This difference is believed to be due to the solubilization and activity of proteases present in the substrate and derived from either feed ingredients or animals. S1 protease from Saccharothrix australiensis numerically increased the level of soluble primary amine in three assays compared to a blank incubated for 1-3 hours without protease.

Claims (22)

(A) at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% relative to the mature polypeptide of SEQ ID NO: 2 A polypeptide having a sequence identity of at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%;
(B) at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89% relative to the mature polypeptide encoding the sequence of SEQ ID NO: 1 Having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity A polypeptide encoded by the polynucleotide;
(C) a variant of the mature polypeptide of SEQ ID NO: 2 comprising substitutions, deletions and / or insertions at one or more (eg several) positions; and (d) having protease activity (a) An isolated polypeptide having protease activity selected from the group consisting of fragments of the polypeptide of (b) or (c).   2. The polypeptide of claim 1 comprising or consisting of SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO: 2.   The polypeptide according to claim 2, wherein the mature polypeptide is amino acids 189 to 374 of SEQ ID NO: 2.   The polypeptide according to any one of claims 1 to 3, which is a variant of the mature polypeptide of SEQ ID NO: 2 comprising substitutions, deletions and / or insertions at one or more positions.   A composition comprising the polypeptide according to any one of claims 1 to 4.   6. A composition according to claim 5, which is a detergent composition such as a laundry or automatic dishwashing composition.   One or more additional selected from protease, amylase, lipase, cutinase, cellulase, endoglucanase, xyloglucanase, pectinase, pectin lyase, xanthanase, peroxidase, haloperoxygenase, catalase, mannanase or any mixture thereof The composition of claim 6 further comprising:   The composition according to claim 6 or 7, comprising one or more components selected from a surfactant, a builder and the like.   An isolated polynucleotide encoding the polypeptide of any one of claims 1-4.   10. A nucleic acid construct or expression vector comprising the polynucleotide of claim 9, operably linked to one or more control sequences that produce said polypeptide in an expression host.   10. A recombinant host cell comprising the polynucleotide of claim 9 operably linked to one or more control sequences that produce said polypeptide. And (a) culturing cells producing said polypeptide in wild-type form under conditions that allow production of said polypeptide; and (b) recovering said polypeptide. A method for producing the polypeptide according to any one of the above. A polypeptide having protease activity, comprising: (a) culturing the host cell of claim 11 under conditions capable of producing the polypeptide; and (b) recovering the polypeptide. How to generate.   A method for improving the nutritional value of animal feed, the method comprising adding at least one protease according to any one of claims 1 to 4 to the feed. a. At least one protease according to any one of claims 1 to 4; and b. At least one fat-soluble vitamin, and / or c. At least one water-soluble vitamin, and / or d. An animal feed additive comprising at least one trace metal.   16. The animal feed additive according to claim 15, further comprising amylase; phytase; xylanase; galactanase; alpha galactosidase; protease, phospholipase; and / or beta glucanase.   An animal feed having a crude protein content of 50-800 g / kg and comprising at least one protease according to any one of claims 1-4.   A method for processing a protein comprising the step of adding at least one protease according to any one of claims 1 to 4 to at least one protein or protein source.   The method of claim 18, wherein soybean is included in the at least one protein source.   Use of at least one protease according to any one of claims 1 to 4 in a detergent.   The animal feed composition containing the polypeptide of any one of Claims 1-4.   12. The animal feed composition of claim 11, further comprising one or more amylases; phytases; xylanases; galactanases; alpha galactosidases; proteases, phospholipases, beta glucanases, or mixtures thereof.